Dual-aav vector-based systems and methods for delivering oversized genes to mammalian cells

ABSTRACT

Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of PCT Intl. Patent Appl. No. PCT/US2012/065645 filed Nov. 16, 2012 (nationalized; Atty. Dkt. No. 36689.353), which claimed priority to U.S. Provisional Patent Appl. No. 61/560,437, filed Nov. 16, 2011 (expired); the contents of each of which is hereby incorporated in its entirety by express reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbers EY00331 and EY021721 awarded by the National Institutes of Health. The government has certain rights in the invention.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the fields of molecular biology and virology, and in particular, to the development of gene delivery vehicles. Disclosed are improved rAAV dual-vector systems, and compositions useful in delivering a variety of nucleic acid segments, including those encoding therapeutic proteins polypeptides, peptides, antisense oligonucleotides, or ribozyme constructs to selected host cells for use in various gene-therapy regimens. Methods are also provided for preparing and using these modified rAAV-dual-vector based systems in a variety of viral-based gene therapies, and in particular, for the treatment and/or amelioration of symptoms of Myosin VII-deficiency, including, without limitation, the treatment of human Usher syndrome.

Description of Related Art

As has been established by a multitude of successful proof-of-concept studies, and various clinical trials, recombinant AAV has emerged as the most optimal gene delivery vehicle to treat retinal disease. However, one limitation of AAV is its relatively small DNA packaging capacity—approximately 4.7 kilobases (KB). Thus, standard AAV vector systems are unsuitable for addressing diseases in which large genes are mutated or otherwise dysfunctional. An example of such a disease is Usher syndrome.

The most common form of Usher syndrome, USH1B, is a severe autosomal-recessive, deaf-blindness disorder caused by mutations in the myosin VIIa gene. Blindness occurs from a progressive retinal degeneration that begins after deafness, and after development of the retina. MYO7a protein is expressed in photoreceptors and retinal pigment epithelium (RPE), and is involved in opsin transport through photoreceptor cilia and the movement of RPE melanosomes.

The coding region for the human Myosin VII protein (MYO7a), however, is 6534 or 6648 nucleotides in length (depending on the allelic variant), making traditional AAV vector systems unsuitable for gene therapy of USH1B.

Previously, Allocca et al. (2008) published intriguing results suggesting that AAV5 serotype vectors were capable of packaging genomes of up to 8.9 KB in size, and that these vectors expressed full-length proteins when delivered in vivo. In this study, the authors expressed full-length MYO7A protein from an AAV5 vector containing the CMV promoter driving hMyo7a. Subsequent studies, carried out to directly validate the Allocca et al. findings were simultaneously published by three independent groups (Lai et al., 2010; Dong et al., 2010; Wu et al., 2010), and their publication was accompanied by an expert commentary (See, Hirsch et al., 2010). While all three studies confirmed that these ‘oversized’ AAV5 vectors did indeed drive full-length protein expression, the genetic content of each vector capsid was found to be limited only to ˜5 KB of DNA, and not the 8.7 KB originally reported by Allocca et al. (2008). These vector capsids were shown to contain a “heterogeneous mixture” of truncated vector genomes (e.g., the 5′-end of the gene, the 3′-end of the gene, or a mixture of the two, with an internal sequence deletion) (Lai et al., 2010; Dong et al., 2010; Wu et al., 2010). Additionally, these oversized/heterogeneous vectors exhibited poor packaging efficiency (i.e., low-vector titers) and low transduction efficiency when compared to matched reporter vectors of standard size (<5 KB) (Wu et al., 2010).

Using this ‘heterogeneous’ system, vectors containing portions of the MYO7A transgene were packaged, however, and proof-of-concept results were demonstrated in the shaker-1 mouse model of USH1B. The therapeutic results achieved with the heterogeneous AAV-hMyo7a vectors were comparable to previous gene replacement results using a Lentivirus-based hMyo7a vector (Hashimoto et al., 2007).

This Lentivirus-Myo7a vector is under development by Oxford BioMedica in collaboration with Sanofi-Aventis for a phase I/II clinical trial of USH1B, marketed under the name UshStat® LentiVector®. Lentivirus is regarded as a vector platform that is not well-suited for infecting post-mitotic (i.e., non-dividing) cells. Furthermore, although the vector is suitable for transducing RPE, many studies have shown it to be ineffective at transducing adult photoreceptors. Even though MYO7A is expressed in both cell types, UshStat® may only be effective at rescuing the RPE phenotype. A study showed that PRs are actually the initial site of disease, so not targeting this cell type effectively may result in zero therapy, although it remains to be seen in the human clinical trial.

Because of the excellent safety profile and encouraging reports of efficacy in the AAV gene therapy trials for LCA2/RPE65, there has been continuing interest in creating an AAV-based system for treating USH1B patients. However, the current AAV vector for MYO7A, as previously mentioned, is heterogeneous; it is manufactured and purified as a single-virus preparation containing a mixture of viral payloads. This fact, unfortunately, makes it virtually impossible to characterize the vector fully—a requirement for government regulatory review and approval. In order to address this concern directly (i.e., that the vector genome of any AAV-based vectors for USH1B must be fully characterized before gaining Food and Drug Administration (FDA) approval, the inventors have developed an AAV dual-vector-based system to facilitate gene therapy approaches for treating USH1B.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods for gene therapy of diseases, such as Usher syndrome 1B (USH1B). The inventors have characterized dual vector platforms described herein as to transcript fidelity, and have shown and that mRNA arising from the system is 100% accurate relative to what would be predicted by correct homologous recombination on the front and back vector pairs, making them useful as gene therapy delivery vector systems.

One aspect of the invention concerns a dual AAV vector system that permits expression of full-length proteins, whose coding sequence exceeds the polynucleotide packaging capacity of an individual AAV vector.

In one embodiment, a vector system of the invention comprises:

(i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end of the polynucleotide, and a suitable promoter followed by a partial coding sequence that encodes an N-terminal part of a selected full-length polypeptide; and

(ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end of the polynucleotide, and a partial coding sequence that encodes a C-terminal part of the selected full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence.

The coding sequence in the first and second AAV vectors comprises sequence that overlaps, such that part of the coding sequence present at the 3′-end of the coding sequence of the first vector is identical or substantially identical with part of the coding sequence present at the 5′-end of the coding sequence of the second vector. In an illustrative embodiment, the polypeptide encoded is a wild type or biologically-functional human myosin VIIa polypeptide (hmyo7a).

In another embodiment, a vector system of the invention includes:

(i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal part of a selected full-length polypeptide followed by a splice donor site for an intron and an intron; and

(ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal part of the selected full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence.

The intron sequence in the first and second AAV vectors includes a sequence that overlaps, such that all or part of the intron sequence present at the 3′-end of the coding sequence of the first vector is identical, or substantially identical, with all or part of the intron sequence present at the 5′-end of the coding sequence of the second vector. In one embodiment, the intron is intron 23 of the hmyo7a gene. In a specific embodiment, the polypeptide encoded is a wild type (i.e., functional) human myosin VIIa polypeptide, and the intron is the full intron 23 of the hmyo7a gene.

BRIEF DESCRIPTION OF THE DRAWINGS

For promoting an understanding of the principles of the invention, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will, nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the invention relates.

The following drawings form part of the present specification and are included to demonstrate certain aspects of the present invention. The application contains at least one drawing that is executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

FIG. 1 shows the formation of complete gene cassette from dual AAV vectors via homologous recombination;

FIG. 2 shows a schematic of the two vector components that make up Dual-Vector System 1 in accordance with one aspect of the present invention;

FIG. 3 shows a schematic of the two vector components that make up Dual-Vector System 2 in accordance with one aspect of the present invention. Native hMyo7a intron 23 in shown in light green; splice donor and splice acceptor sequence are shown in dark green;

FIG. 4 shows the schematic of the two vector components that make up Dual-Vector System 3 in accordance with one aspect of the present invention. This is an exemplary standard transplicing dual vector pairs with the “intron” in this case referring to the alkaline phosphatase splice donor and acceptor sites. Synthetic alkaline phosphatase (AP) intron is shown in light blue; AP splice donor and splice acceptor sequences are shown in dark blue;

FIG. 5 shows an immunoblot to detect the presence of MYO7A in infected or transfected HEK293 cells. Heterogeneous vectors (described in introduction) are compared to all three dual-vector systems. Dual vectors were packaged either in AAV2 or in AAV2 (triple mutant) capsids. The triple mutant contains three tyrosine-to-phenylalanine mutations on the capsid surface. For all three dual-vector systems, infections were performed with either a) the front-half (N-terminal) and back-half (C-terminal) vectors; or b) the front-half vectors alone (to confirm the presence or absence of a truncated protein product expressed from the promoter-containing N-terminal vectors);

FIG. 6A and FIG. 6B show immunoblot to detect the presence of myo7A in HEK293 cells infected with Dual-Vector System 1. Results are presented as a time course from 3-7 days post infection (lanes 3-7) and are compared to cells transfected with myo7a plasmid (lane 1) and uninfected control (lane 2). The area inside the white box shown in FIG. 6A is magnified and presented at higher contrast in FIG. 6B. Starting at 3-days' post-infection, full-length human MYO7a protein was visible, with peak expression occurring around day 5;

FIG. 7A and FIG. 7B show retinas from untreated mice and mice treated subretinally with Dual-Vector System 1. Immunohistochemistry (IHC) was performed using an antibody directed against MYO7A. The ‘green’ is the stain of MYO7A and ‘blue’ corresponds to nuclear, DAPI stain;

FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D show differences in RPE melanosome localization in wild type vs. shaker-1 mice. In wild type mice, RPE melanosome apically migrate towards photoreceptor outer segments (FIG. 8A) whereas this phenomenon fails to occur in mice lacking Myo7a (shaker-1), as seen in (FIG. 8B). To the right is a high magnification image of single RPE cells from either a wild type (FIG. 8C) or shaker-1 (FIG. 8D) mouse showing this phenomenon up close;

FIG. 9A, FIG. 9B, and FIG. 9C show apical migration of RPE melanosomes is restored in shaker-1 mice injected with Dual-Vector System 1. Electron microscopy reveals that melanosomes of untreated shaker-1 mice do not apically migrate (FIG. 9A). In shaker-1 mice injected with Dual-Vector System 1 (packaged in AAV2), RPE melanosomes migrate apically towards photoreceptors, which can be seen here in both low- and high-magnification images (FIG. 9B and FIG. 9C);

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E, and FIG. 10F illustrate the expression of MYO7A from single AAV2 and AAV5 vectors in cultured cells. FIG. 10A is a diagram of the viral vector encoding human MYO7A cDNA. FIG. 10B is a western blot of WT eyecup (lane 1), primary RPE cultures derived from Myo7a-null mice and infected with AAV2-MYO7A (lane 2) or AAV5-MYO7A (lane 3), or not infected (lane 4), and primary RPE cultures derived from Myo7a^(+/−) mice (lane 5). All lanes were immunolabeled with antibodies against actin (as a loading indicator of relative protein loading) and MYO7A. FIG. 10C, FIG. 10D, FIG. 10E, and FIG. 10F are immunofluorescence images of primary RPE cell cultures. Cells derived from Myo7a-null mice that were not infected (FIG. 10C), from Myo7a^(+/−) mice (FIG. 10D), or from Myo7a-null mice infected with either 1×AAV2-MYO7A (FIG. 10E) or 1×AAV5-MYO7A (FIG. 10F). Scale=10 μm;

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F, FIG. 11F-1, FIG. 11G, FIG. 11G-1, FIG. 11H, FIG. 11I, FIG. 11J, FIG. 11K, FIG. 11L, and FIG. 11M show the expression of MYO7A from single AAV2 and AAV5 vectors in vivo. FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E show EM images of MYO7A immunogold labelling of the connecting cilium and pericilium from rod photoreceptors in a Myo7a-null retina. FIG. 11A is a longitudinal section from an untreated Myo7a-null retina (background label only). FIG. 11B and FIG. 11C are longitudinal sections from Myo7a-null retinas treated with AAV2-MYO7A (FIG. 11B) or AAV5-MYO7A (FIG. 11C). Scale=50 nm. FIG. 11D and FIG. 11E are transverse sections of connecting cilia from rod photoreceptors in Myo7a-null retinas treated with AAV2-MYO7A (FIG. 11D) or AAV5-MYO7A (FIG. 11E). Scale=50 nm. FIG. 11F and FIG. 11G show EM images of RPE cells from Myo7a-null retinas treated with AAV2-MYO7A (FIG. 11F) or AAV5-MYO7A (FIG. 11G). Scale=500 nm. BM=Bruch's membrane, AP=apical processes. Areas indicated by rectangles are enlarged in FIG. 11F-1 and FIG. 11G-1 to show MYO7A immunogold labeling (indicated by circles). Scale=50 nm. FIG. 11H and FIG. 11I show EM image of a longitudinal section of the connecting cilium and pericilium from a rod (FIG. 11H) and a cone (FIG. 11I) photoreceptor in a Myo7a-null retina, treated with AAV2-MYO7A. The section was double-labeled with MYO7A (12-nm gold) and rod opsin (15-nm gold) antibodies. Rod outer segments were labeled with the opsin antibody, while cones were identified by lack of rod opsin labeling in their outer segments. The sections show just the base of the outer segments. Nearly all the label in the connecting cilium is MYO7A, even in the rod. Scale=50 nm. FIG. 11J, FIG. 11K, FIG. 11L, and FIG. 11M are bar graphs indicating MYO7A immunogold particle density in the rod photoreceptor cilium and pericilium (FIG. 11J and FIG. 11K) and in the RPE (FIG. 11L and FIG. 11M), following treatment with AAV2-MYO7A (FIG. 11J and FIG. 11L) or AAV5-MYO7A (FIG. 11K and FIG. 11M) of different concentrations. n=3 animals per condition. Bars indicate SEM;

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, and FIG. 12F show correction of melanosome localization, following subretinal injections with AAV2-MYO7A or AAV5-MYO7A. Light micrographs showing the presence of melanosomes in the apical processes of the RPE in a WT retina (FIG. 12A) and retinas injected with AAV2-MYO7A (FIG. 12B) or AAV5-MYO7A (FIG. 12C). Further away from the injection site (FIG. 12D), melanosomes are present in the apical processes of some RPE cells, but not in others (arrows indicate apical melanosomes; white lines indicate regions where melanosomes are absent from the apical processes). FIG. 12E illustrates a region distant from injection site, where all RPE cells lacked melanosomes in their apical processes. Brackets on left side indicate RPE apical processes. Scale=25 μm. FIG. 12F is a diagram of an eyecup, indicating the relative locations of the images shown in FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, and FIG. 12E. Arrow indicates the site of injection; ONH indicates the optic nerve head;

FIG. 13 shows the correction of abnormal levels of opsin in the connecting cilium and pericilium of rod photoreceptors, following subretinal injections with AAV2-MYO7A or AAV5-MYO7A. The bar graph shows opsin immunogold gold particle density along the length of the connecting cilium. Ultrathin sections of retinas from Myo7a-null and WT mice were stained with rod opsin antibody. The Myo7a-null retinas had been untreated, or treated with either 1× or 1:100 AAV2-MYO7A or AAV5-MYO7A. n=3 animals per condition. Bars indicate SEM;

FIG. 14A-1, FIG. 14A-2, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F, and FIG. 14G show the expression of MYO7A from the overlapping AAV2-MYO7A dual vectors. FIG. 14A-1 and FIG. 14A-2 illustrate a diagram of the overlapping AAV2-MYO7A dual vectors. The overlapping region contains 1365 bases. FIG. 14B is a Western blot of proteins from primary RPE cultures derived from Myo7a-null mice and not infected (lane 1), or infected with AAV2-MYO7A(dual) (lane 2); primary RPE cultures derived from Myo7a^(+/−) mice (lane 3); WT eyecup (lane 4); HEK293A cells transfected with pTR-smCBA-MYO7A (lane 5). All lanes were immunolabeled with anti-MYO7A and anti-actin. FIG. 14C, FIG. 14D, FIG. 14E, and FIG. 14F show immunofluorescence of cultured RPE cells transduced with AAV2-MYO7A(dual). FIG. 14C, FIG. 14D, and FIG. 14E show primary RPE cultures derived from Myo7a-null mice and ARPE19 (FIG. 14F) cells. Scale=10 μm. FIG. 14G is a bar graph indicating the distribution of MYO7A immunogold particle density among RPE cells from retinas of Myo7a-null mice, injected with AAV2-MYO7A(dual). n=3 animals;

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, FIG. 15F, and FIG. 15G illustrate correction of mutant phenotypes, following subretinal injections with AAV2-MYO7A(dual). FIG. 15A is the results of light microscopy of a semi-thin section from a treated Myo7a-null mouse retina. The region shown is near the injection site. Arrows indicate melanosomes in the apical processes. White lines indicate cells that still show the Myo7a-null phenotype, with an absence of melanosomes in the apical processes. Scale=50 μm. FIG. 15B is a low-magnification immunoEM image of RPE from a retina treated with AAV2-MYO7A(dual). As in FIG. 15A, the white line indicated a region that still showed the Myo7a-null phenotype. Rectangle ‘c’, includes melanosomes in the apical region, indicating a corrected RPE cell. Scale=500 nm. FIG. 15C, FIG. 15D, and FIG. 15E show higher-magnification images of regions outlined by the rectangles shown in FIG. 15B. MYO7A immunogold particles were indicated by circles. Scale=50 nm. FIG. 15F is a bar graph illustrating MYO7A immunogold particle density measured in RPE cells from Myo7a-null retinas, WT retinas, or from Myo7a-null retinas treated with AAV2-MYO7A(dual) and determined to be corrected or not corrected by the location of their apical melanosomes. n=3 animals per condition. Bars indicate SEM. FIG. 15G is an immunoEM image of a rod photoreceptor cilium double-labeled with antibodies against MYO7A (small gold particles) and against rod opsin (large gold particles). MYO7A labeling is associated with the connecting cilium and periciliary membrane, indicating expression and correct localization of MYO7A. While this region is devoid of opsin labeling, which is restricted to the disk membranes, it is consistent with the wild type (WT) phenotype, thus indicating correction of the mutant phenotype. Scale=300 nm;

FIG. 16 shows the validation of dual AAV vectors for delivery of full-length MYO7A in vivo. Immunoblot showing expression of MYO7A (green) in retinas of wild type (C57BL/6) mice (lane 1), heterozygous shaker-1^(+/) mice (lane 2) and shaker-1^(−/−) mice injected with ‘simple overlap’ Myo7a vectors packaged in AAV8(733) vectors. Both N-terminal and C-terminal vectors of the ‘simple overlap’ system were injected at a concentration of 3×10¹⁰ vector genomes/μL. Dual AAV vectors mediated expression of a MYO7A that was identical in size to that found in WT and shaker-1^(+/−) mice. β-actin (visualized here in red) was used as a loading control to validate that equal amounts of protein were loaded in each well;

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F show AAV-mediated MYO7A expression in ARPE-19. Cells were transduced with 1×AAV2-MYO7A (FIG. 17A), AAV5-MYO7A (FIG. 17B), 1/100 dilutions thereof (FIG. 17C and FIG. 17D) and AAV2-MYO7A (dual) (FIG. 17F). Non-transduced cells were used as a control (FIG. 17E); Red, MYO7A; Blue, DAPI. Scale=10 μm;

FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show MYO7A expression in the connecting cilium and pericilium of rod photoreceptors from Myo7a-null retinas injected with diluted AAV2-MYO7A (FIG. 18a and FIG. 18B) or AAV5-MYO7A (FIG. 18C and FIG. 18D); (FIG. 18A and FIG. 18C) 1:10, (FIG. 18B and FIG. 18D) 1:100. Scale=200 nm;

FIG. 19 shows the structural preservation of injected Myo7a-null retinas. Light microscopy of the photoreceptor layer 3 weeks after injection with 10×AAV5-MYO7A. Scale=15 μm;

FIG. 20 shows structural preservation of injected Myo7a-null retinas. Light microscopy of photoreceptor layer 3 months after injection with 1×AAV2-MYO7A. Scale=10 μm;

FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D show correction of abnormal levels of opsin in the connecting cilium and pericilium of rod photoreceptors following subretinal injections with AAV2-MYO7A or AAV5-MYO7A. ImmunoEMs from WT retina (FIG. 21A), Myo7a-null retinas treated with 1×AAV2-MYO7A (FIG. 21B) or 1×AAV5-MYO7A (FIG. 21C), and from an untreated Myo7a-null retina (FIG. 21D) labeled with anti-rod opsin and 12-nm gold-conjugated secondary antibody. Scale=200 nm;

FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D, and FIG. 22E show a schematic representation of the dual-AAV-vector pairs created for this study. FIG. 22A is a fragmented vector (fAAV). FIG. 22B shows simple overlap: the 1365-bp shared between the two vectors is shaded gray. FIG. 22C is a trans-splicing vector. FIG. 22D shows an AP hybrid vector: the 270-bp element shared between the two vectors is marked with diagonal gradient shading (⅓ head as described by Ghosh et al., 2011). FIG. 22 shows the native intron hybrid vectors utilizing the natural intron 23 of MYO7A sharing a 250-bp overlapping sequence. 3′MYO7A is the 3′-portion of MYO7A; 5′MYO7A is the 5′-portion of MYO7A; AAV is adeno-associated virus; AP is alkaline phosphatase; intron=intron 23 of MYO7A; pA=polyadenylation signal; SA=splice-acceptor site; SD=splice-donor site; smCBA=cytomegalovirus immediate early/chicken β-actin chimeric promoter;

FIG. 23A, FIG. 23B, and FIG. 23C show human embryonic kidney (HEK293) cells express human MYO7A after infection with simple overlap vectors (MOI of 10,000 for both vectors) packaged in AAV2(tripleY-F). Equal amounts of protein were separated on 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and stained for MYO7A. FIG. 23A shows HEK293 cells infected with AAV2(tripleY-F) at MOIs of 10,000, 2000, and 400 FIG. 23B is a time-course assay of MYO7A expressed in HEK293 cells. Cells were harvested 3-7 days after infection. In FIG. 23C, HEK293 cells were infected with AAV2 dual vectors; MOI=multiplicity of infection; T=HEK293 cells transfected with full-length MYO7A plasmid; U=untreated HEK293 cells;

FIG. 24 shows the comparison of AAV2 and AAV2(tripleY→F mutant capsid)-based vectors in HEK293 cells. Cells were infected with single fAAV, AP hybrid, and simple overlap MYO7A dual-vector platforms packaged in AAV2 or AAV2(tripleY→F mutant capsid) at an MOI of 10,000. HEK293 cells transfected with MYO7A plasmid were used as a positive control;

FIG. 25A, FIG. 25B, and FIG. 25C show human MYO7A expressed in HEK293 cells. Cells were infected with AAV2-based vector platforms. For each of the dual-vector systems, the corresponding 5′ and 3′ vectors (or the 5′ vector alone) were used for infection. HEK293 cells transfected with MYO7A plasmid were used as a positive control. Cells were infected with the MYO7A dual-vector pairs at an MOI of 10,000 for each vector. Protein samples were analyzed on Western blot with an antibody against MYO7A (FIG. 25A). Each platform's relative ability to promote reconstitution was compared by quantifying the amount of 5′ vector-mediated truncated protein product in the presence or absence of the respective 3′ vector (FIG. 25B). Full-length MYO7A expression mediated by dual vectors was quantified relative to transfection control (FIG. 25C);

FIG. 26A, FIG. 26B, and FIG. 26C show the characterization of MYO7A dual-vectors' restoration of coding sequence. The experimental plan is shown in FIG. 26A. HEK293 cells were infected with AAV2-based dual-vector platforms, RNA was extracted, and gene-specific primers amplified the sequences using PCT. Control digests with BglII (B) and PpuMI (P) revealed the predicted banding pattern shown in FIG. 26B. Undigested (U) PCR product is shown as control and a DNA size marker for reference (M). Separately, products were digested with KpnI and AgeI, and then cloned into pUC57 for sequencing of the entire overlap region FIG. 26C. Ten clones per vector platform were analyzed. M13 forward- and reverse-primers specific for the subclone vector were used to obtain sense and antisense reads (each ˜1000 bp) resulting in ˜140 bp for which the sense and antisense reads overlapped. PCR=polymerase chain reaction and

FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D, FIG. 27E, FIG. 27F, FIG. 27G, and FIG. 27H show the dual vector-mediated MYO7A(HA) expression in vivo. C57BL/6J mice were injected subretinally with AAV2-based dual vectors containing a C′ terminal HA tag. Retinal protein expression was analyzed four weeks later by immunohistochemistry and western blot. Ten-micron frozen retinal cross sections were imaged at 10× (FIG. 27A, FIG. 27C, and FIG. 27E) and 60× (FIG. 27B, FIG. 27D, and FIG. 27F). Equal amounts of protein were separated on a 4-15% polyacrylamide gel and stained with an HA antibody (FIG. 27H). For comparison, endogenous MYO7A from C57BL/6J retina (FIG. 27G) was probed with an antibody against MYO7A to confirm that HA-tagged MYO7A migrated at the appropriate size. RPE—retinal pigment epithelium, IS—inner segments, OS—outer segments, ONL—outer nuclear layer, INL—inner nuclear layer, GCL—ganglion cell layer, PR—photoreceptors.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of an “hMyo7a coding overlap vector A” of the subject invention;

SEQ ID NO:2 is the nucleotide sequence of an “hMyo7a coding overlap vector B” of the subject invention;

SEQ ID NO:3 is the nucleotide sequence of an “hMyo7a intron 23 splicing vector A” of the subject invention;

SEQ ID NO:4 is the nucleotide sequence of an “hMyo7a intron 23 splicing vector B” of the subject invention;

SEQ ID NO:5 is a nucleotide sequence encoding a human myosin VIIa polypeptide (protein coding sequence is nucleotides 273-6920);

SEQ ID NO:6 is the amino acid sequence of the human myosin VIIa polypeptide encoded by nucleotides 273-6920 of SEQ ID NO:5;

SEQ ID NO:7 is a nucleotide sequence that encodes a human myosin VIIa polypeptide;

SEQ ID NO:8 is an amino acid sequence of a human myosin VIIa polypeptide (isoform 2);

SEQ ID NO:9 is a synthetic oligonucleotide sequence, designated herein as P1, useful in accordance with one aspect of the present invention;

SEQ ID NO:10 is a synthetic oligonucleotide sequence, designated herein as P2, useful in accordance with one aspect of the present invention;

SEQ ID NO:11 is a synthetic oligonucleotide sequence, designated herein as P3, useful in accordance with one aspect of the present invention;

SEQ ID NO:12 is a synthetic oligonucleotide sequence, designated herein as P4, useful in accordance with one aspect of the present invention;

SEQ ID NO:13 is a synthetic oligonucleotide sequence, designated herein as P5, useful in accordance with one aspect of the present invention;

SEQ ID NO:14 is a synthetic oligonucleotide sequence, designated herein as P6, useful in accordance with one aspect of the present invention;

SEQ ID NO:15 is a synthetic oligonucleotide sequence, designated herein as P7, useful in accordance with one aspect of the present invention;

SEQ ID NO:16 is a synthetic oligonucleotide sequence, designated herein as P8, useful in accordance with one aspect of the present invention;

SEQ ID NO:17 is a synthetic oligonucleotide sequence, designated herein as P9, useful in accordance with one aspect of the present invention;

SEQ ID NO:18 is a synthetic oligonucleotide sequence, designated herein as P10, useful in accordance with one aspect of the present invention;

SEQ ID NO:19 is a synthetic oligonucleotide sequence, designated herein as P11, useful in accordance with one aspect of the present invention;

SEQ ID NO:20 is a synthetic oligonucleotide sequence, designated herein as P12, useful in accordance with one aspect of the present invention;

SEQ ID NO:21 is a synthetic oligonucleotide sequence, designated herein as P13, useful in accordance with one aspect of the present invention;

SEQ ID NO:22 is a synthetic oligonucleotide sequence, designated herein as P14, useful in accordance with one aspect of the present invention;

SEQ ID NO:23 is a synthetic oligonucleotide sequence, designated herein as P15, useful in accordance with one aspect of the present invention;

SEQ ID NO:24 is a synthetic oligonucleotide sequence, designated herein as P16, useful in accordance with one aspect of the present invention;

SEQ ID NO:25 is a synthetic oligonucleotide sequence, designated herein as P17, useful in accordance with one aspect of the present invention;

SEQ ID NO:26 is a synthetic oligonucleotide sequence, designated herein as P18, useful in accordance with one aspect of the present invention;

SEQ ID NO:27 is a synthetic oligonucleotide sequence, designated herein as P19, useful in accordance with one aspect of the present invention;

SEQ ID NO:28 is a synthetic oligonucleotide sequence, designated herein as P20, useful in accordance with one aspect of the present invention;

SEQ ID NO:29 is a synthetic oligonucleotide sequence, designated herein as P21, useful in accordance with one aspect of the present invention; and

SEQ ID NO:30 is a synthetic oligonucleotide sequence, designated herein as P22, useful in accordance with one aspect of the present invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

The subject invention concerns materials and methods for genetic therapy of diseases and conditions, such as Usher syndrome 1B (USH1B). One aspect of the invention concerns AAV-based dual-vector systems that provide for expression of full-length proteins whose coding sequence exceeds the polynucleotide packaging capacity of individual AAV vector. In one embodiment, a vector system of the invention includes:

i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end (5′ and 3′ end) of the polynucleotide, and between the inverted terminal repeats a suitable promoter followed by (i.e., 3′ to the promoter) a partial coding sequence that encodes an N-terminal part of a selected full-length polypeptide, and

ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end (i.e., the 5′- and 3′-ends) of the polynucleotide, and between the inverted terminal repeats a partial coding sequence that encodes a C-terminal portion of the selected full-length polypeptide, and optionally followed by a polyadenylation (pA) sequence. The coding sequences in the first and second vectors when combined encode the selected full-length polypeptide, or a functional fragment or variant thereof. The polypeptide encoding sequence in the first and second AAV vectors comprises sequence that overlaps.

In other words, a portion of the coding sequence present at the 3′-end of the coding sequence of the first vector is identical or substantially identical with a portion of the coding sequence present at the 5′-end of the coding sequence of the second vector. In one embodiment, the sequence overlap between the first and second AAV vectors is between about 500 and about 3000 nucleotides; between about 1000 and about 2000 nucleotides; between about 1200 and about 1800 nucleotides; or between about 1300 and about 1400 nucleotides.

In a specific embodiment, the sequence overlap is about 1350 nucleotides. In an exemplified embodiment, the sequence overlap is approximately 1365 nucleotides. In one embodiment, the polypeptide encoded is wild type or functional human myosin VIIa (hmyo7a). Amino acid sequences of wild type and functional hmyo7a polypeptides, and polynucleotides encoding them, are known in the art (see, for example, GenBank accession numbers NP_000251 and U39226.1). In one embodiment, an hmyo7a polypeptide comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:8, or a functional fragment or a variant thereof. In one embodiment, the hmyo7a polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:7. Other polypeptides contemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot 075445). In an exemplified embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:1, or a functional fragment and/or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:2, or a functional fragment and/or variant thereof. In one embodiment, a construct or vector of the invention is administered by parenteral administration, such as intravenous, intramuscular, intraocular, intranasal, etc. In a specific embodiment, a construct or vector is administered by subretinal injection. The construct or vector can be administered in vivo or ex vivo.

In another embodiment, a vector system of the invention comprises i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end (i.e., the 5′-end and the 3′-end) of the polynucleotide, and between the inverted terminal repeats a suitable promoter followed by (i.e., 3′ to the promoter) a partial coding sequence that encodes an N-terminal part of a selected full-length polypeptide followed by a splice donor site and an intron, and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end (5′-end and 3′-end) of the polynucleotide, and between the inverted terminal repeats an intron and a splice acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal part of the selected full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence.

The coding sequences in the first and second vectors when combined encode the selected full-length polypeptide, or a functional fragment or variant thereof. The intron sequence in the first and second AAV vectors comprises sequence that overlaps. In other words, all or part of the intron sequence present at the 3′-end of the coding sequence of the first vector is identical or substantially identical with all or part of the intron sequence present at the 5′-end of the coding sequence of the second vector. In one embodiment, intron sequence overlap between the first and second AAV vectors is several hundred nucleotides in length. In a specific embodiment, the intron sequence overlap is about 50 to about 500 nucleotides or so in length; alternatively between about 200 and about 300 nucleotides or so in length. In one embodiment, the intron sequence utilized in the vector system of the invention is a sequence of an intron naturally present in the genomic sequence of a gene encoding the selected polypeptide. In one embodiment, the intron is intron 23 of the hmyo7a gene. In a specific embodiment, the polypeptide encoded is hmyo7a and the intron is the full intron 23 of the hmyo7a gene. In an exemplified embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:3, or a functional fragment and/or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:4, or a functional fragment and/or variant thereof. In another embodiment, the intron sequence utilized in the vector system of the invention is a sequence of an intron that is not naturally present in the genomic sequence of a gene encoding the selected polypeptide. In a specific embodiment, the intron is a synthetic alkaline phosphatase (AP) intron. The intron sequences utilized in the vector system of the present invention can comprise splice donor and splice acceptor sequences. In one embodiment, a construct or vector of the invention is administered by parenteral administration, such as intravenous, intramuscular, intraocular, intranasal, etc. In a specific embodiment, a construct or vector is administered by subretinal injection. The construct or vector can be administered in vivo or ex vivo.

The inverted terminal repeat (ITR) sequences used in an AAV vector system of the present invention can be any AAV ITR. The ITRs used in an AAV vector can be the same or different. In particular embodiments, the ITR may be obtained from an AAV serotype 2 (AAV2) or an AAV serotype 5 (AAV5). An AAV vector of the invention can comprise different AAV ITRs. For example, a vector may comprise an ITR of AAV2 and an ITR of AAV5. AAV ITR sequences are well known in the art (see, for example, GenBank Accession Nos. AF043303.1; NC_001401.2; J01901.1; JN898962.1; K01624.1; and K01625.1).

The AAV dual-vector systems disclosed herein are able to efficiently express a therapeutic gene that is larger than what may ordinarily be packaged within a single AAV vector.

The subject invention also concerns a virus or virion comprising a polynucleotide, expression construct, or vector construct of the invention. In one embodiment, the virus or virion is an AAV virus. Methods for preparing viruses and virions comprising a heterologous polynucleotide or construct are known in the art. In the case of AAV, cells can be co-infected or transfected with adenovirus or polynucleotide constructs comprising adenovirus genes suitable for AAV helper function. Examples of materials and methods are described, for example, in U.S. Pat. Nos. 8,137,962 and 6,967,018 (each of which is specifically incorporated herein, by express reference thereto).

An AAV virus or AAV vector of the invention can be of any AAV serotype, including, but not limited to, serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV5, AAV10, AAV11, and AAV12. In exemplary embodiments, AAV2 and AAV5 serotype vectors have been utilized.

In one embodiment, the AAV serotype provides for one or more tyrosine to phenylalanine (Y→F) mutations on the capsid surface. In a specific embodiment, the AAV is an AAV8 serotype having a tyrosine-to-phenylalanine (Tyr→Phe) mutation at position 733 (Y733F). In FIG. 5, a triple-mutant vector is also contemplated. It contains tyrosine-to-phenylalanine (Tyr→Phe) mutations at positions Y733F, Y500F, and Y730F, respectively, corresponding to the amino acid sequence of the wild-type AAV8-capsid protein, or in one or more of the amino acids corresponding to those sequences in one or more related AAV capids, including, for example, AAV2, AAV5, and the like.

The subject invention also concerns methods for treating or ameliorating a disease or condition, such as an eye disease, in a human or animal using gene therapy and an AAV-based dual-vector system of the present invention. In one embodiment, a method of the invention comprises administering a vector system of the invention that encodes a polypeptide that provides for treatment or amelioration of the disease or condition. In one embodiment, the vectors of the invention are provided in an AAV virus or virion. The vector system can be administered in vivo or ex vivo. In one embodiment, a vector system of the invention is administered by parenteral administration, such as intravenous, intramuscular, intraocular, intranasal, etc. Administration can be by injection. In a specific embodiment, a vector system of the invention is administered to the human or animal by intraocular subretinal injection. In one embodiment, the disease or condition to be treated is Usher syndrome and the polypeptide provided is a mammalian myosin VIIa protein. In a specific embodiment, the myosin VIIa is a human myosin VIIa polypeptide. In one embodiment, an hmyo7a polypeptide comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:8, or a functional fragment or a variant thereof. In one embodiment, the hmyo7a polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:7.

Other polypeptides contemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot 075445). Dosage regimes and effective amounts to be administered can be determined by ordinarily skilled clinicians. Administration may be in the form of a single dose or multiple doses. General methods for performing gene therapy using polynucleotides, expression constructs, and vectors are known in the art (see, for example, Gene Therapy: Principles and Applications (1999); and U.S. Pat. Nos. 6,461,606; 6,204,251 and 6,106,826, each of which is specifically incorporated herein in its entirety by express reference thereto).

The subject invention also concerns methods for expressing a selected polypeptide in a cell. In one embodiment, the method comprises incorporating in the cell an AAV-based, dual-vector system as disclosed herein, wherein the vector system includes a polynucleotide sequence that encodes a selected polypeptide and of interest, and expressing the polynucleotide sequences in the cell. In certain embodiments, the selected polypeptide may be a polypeptide that is heterologous to the cell. In one embodiment, the cell is a mammalian cell, and preferably, a human cell. In one embodiment, the cell is human a photoreceptor cell, and preferably a human photoreceptor cone cell or a photoreceptor rod cell. In a specific embodiment, the cell expresses a wild type, functional, and/or biologically-active hmyo7a polypeptide that is encoded by a nucleic acid segment present in a vector system as disclosed herein. In one embodiment, the hmyo7a polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:7. Other polypeptides contemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot 075445). The cell can be one that is provided in vivo or in vitro.

The subject invention also concerns one or more mammalian cells that contain one of the AAV-based, dual-vector systems disclosed herein.

In one embodiment, the cell is a photoreceptor cell. In a specific embodiment, the cell is a cone cell; preferably, it is a human cone cell or a human rod cell. Such cells may express one or more nucleotide sequences provided in at least a first AAV-based, dual-vector system of the invention. In a specific embodiment, the cell expresses a wild type, functional, and/or biologically active hmyo7a polypeptide that is encoded by a nucleic acid segment comprised within one or more of the AAV-based vector systems as disclosed herein. In one embodiment, the hmyo7a polypeptide is encoded by the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:7. Other polypeptides contemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot 075445).

Vector systems of the invention can include regulatory elements that are functional in the intended host cell in which the vector is to be expressed. A person of ordinary skill in the art can select regulatory elements for use in appropriate host cells, for example, mammalian or human host cells. Regulatory elements include, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.

A vector of the invention can comprise a promoter sequence operably linked to a nucleotide sequence encoding a desired polypeptide. Promoters contemplated for use in the subject invention include, but are not limited to, cytomegalovirus (CMV) promoter, SV40 promoter, Rous sarcoma virus (RSV) promoter, chimeric CMV/chicken β-actin promoter (CBA) and the truncated form of CBA (smCBA) (see, e.g., Haire et al. 2006 and U.S. Pat. No. 8,298,818, which is specifically incorporated herein in its entirety by express reference thereto). Additional photoreceptor-specific, human rhodopsin kinase (hGRK1) promoter, rod specific IRBP promoter, VMD2 (vitelliform macular dystrophy/Best disease) promoter, and EF1 alpha promoter sequences are also contemplated to be useful in the practice of various aspects of the present invention.

In a specific embodiment, the promoter is a chimeric CMV-β-actin promoter. In one embodiment, the promoter is a tissue-specific promoter that shows selective activity in one or a group of tissues but is less active or not active in other tissue. In one embodiment, the promoter is a photoreceptor-specific promoter. In a further embodiment, the promoter is preferably a cone cell-specific promoter or a rod cell-specific promoter, or any combination thereof. In one embodiment, the promoter is the promoter for human myosin 7a gene. In a further embodiment, the promoter comprises a cone transducin α (TαC) gene-derived promoter. In a specific embodiment, the promoter is a human GNAT2-derived promoter. Other promoters contemplated within the scope of the invention include, without limitation, a rhodopsin promoter, a cGMP-phosphodiesterase β-subunit promoter, a retinitis pigmentosa-specific promoter, an RPE cell-specific promoter [such as a vitelliform macular dystrophy-2 (VMD2) promoter (Best1) (Esumi et al., 2004)], or any combination thereof.

Promoters can be incorporated into a vector using standard techniques known to those of ordinary skill in the molecular biology and/or virology arts. Multiple copies of promoters, and/or multiple distinct promoters can be used in the vectors of the present invention. In one such embodiment, a promoter may be positioned about the same distance from the transcription start site as it is from the transcription start site in its natural genetic environment, although some variation in this distance is permitted, of course, without a substantial decrease in promoter activity. In the practice of the invention, one or more transcription start site(s) are typically included within the disclosed vectors.

The vectors of the present invention may further optionally include one or more transcription termination sequences, one or more translation termination sequences, one or more signal peptide sequences, one or more internal ribosome entry sites (IRES), and/or one or more enhancer elements, or any combination thereof. Transcription termination regions can typically be obtained from the 3′ untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. Signal peptide sequences are amino-terminal peptidic sequences that encode information responsible for the location of an operably-linked polypeptide to one or more post-translational cellular destinations, including, for example, specific organelle compartments, or to the sites of protein synthesis and/or activity, and even to the extracellular environment.

Enhancers—cis-acting regulatory elements that increase gene transcription—may also be included in one of the disclosed AAV-based vector systems. A variety of enhancer elements are known to those of ordinary skill in the relevant arts, and include, without limitation, a CaMV 35S enhancer element, a cytomegalovirus (CMV) early promoter enhancer element, an SV40 enhancer element, as well as combinations and/or derivatives thereof. One or more nucleic acid sequences that direct or regulate polyadenylation of the mRNA encoded by a structural gene of interest, may also be optionally included in one or more of the vectors of the present invention.

The disclosed dual-vector systems may be introduced into one or more selected mammalian cells using any one or more of the methods that are known to those of ordinary skill in the gene therapy and/or viral arts. Such methods include, without limitation, transfection, microinjection, electroporation, lipofection, cell fusion, and calcium phosphate precipitation, as well as biolistic methods. In one embodiment, the vectors of the invention may be introduced in vivo, including, for example, by lipofection (i.e., DNA transfection via liposomes prepared from one or more cationic lipids) (see, for example, Felgner et al., 1987). Synthetic cationic lipids (LIPOFECTIN, Invitrogen Corp., La Jolla, Calif., USA) may be used to prepare liposomes that will encapsulate the vectors to facilitate their introduction into one or more selected cells. A vector system of the invention can also be introduced in vivo as “naked” DNA using methods known to those of ordinary skill in the art.

Polynucleotides described herein can also be defined in terms of more particular identity and/or similarity ranges with those exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or greater as compared to a sequence exemplified herein.

Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and) XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score=100, word-length=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described (Altschul et al., 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and)XBLAST) can be used in accordance with published methods.

The subject invention also contemplates those polynucleotide molecules having sequences that are sufficiently homologous with the polynucleotide sequences of the invention to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis et al., 1982). As used herein, “stringent” conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25° C. below the melting temperature (T_(m)) of the DNA hybrid in 6×SSPE, 5× Denhardt's solution, and 0.1% SDS, containing 0.1 mg/mL of a suitable non-specific denatured DNA. Calculation of the melting temperature may be obtained using the standard formula of Beltz et al., (1983):

T_(m)=81.5° C.+16.6 Log[Na⁺]+0.41(% G+C)−0.61(% formamide)−600/length of duplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 min in 1×SSPE, 0.1% SDS (i.e., a low-stringency wash); and

(2) Once at T_(m)−20° C. for 15 min in 0.2×SSPE, 0.1% SDS (i.e., a moderate-stringency wash).

As used herein, the terms “nucleic acid” and “polynucleotide sequence” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides. The polynucleotide sequences include both full-length sequences, as well as shorter sequences derived from the full-length sequences. It is understood that a particular polynucleotide sequence includes the degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific host cell. The polynucleotide sequences falling within the scope of the subject invention further include sequences that specifically hybridize with the sequences coding for a peptide of the invention. The polynucleotide includes both the sense and antisense strands, either as individual strands or in the duplex.

Fragments and variants of a polynucleotide or polypeptide of the present invention can be generated as described herein and tested for the presence of function using standard techniques known in the art. Thus, an ordinarily skilled artisan can readily prepare and test fragments and variants of a polynucleotide or polypeptide of the invention and determine whether the fragment or variant retains functional activity that is the same or similar to a full-length or a non-variant polynucleotide or polypeptide, such as a myosin VIIa polynucleotide or polypeptide.

As those skilled in the art can readily appreciate, there can be a number of variant sequences of a protein found in nature, in addition to those variants that can be artificially created by the skilled artisan in the lab. The polynucleotides and polypeptides of the subject invention encompasses those specifically exemplified herein, as well as any natural variants thereof, as well as any variants which can be created artificially, so long as those variants retain the desired functional activity.

Also within the scope of the subject invention are polypeptides which have the same amino acid sequences of a polypeptide exemplified herein except for amino acid substitutions, additions, or deletions within the sequence of the polypeptide, as long as these variant polypeptides retain substantially the same relevant functional activity as the polypeptides specifically exemplified herein. For example, conservative amino acid substitutions within a polypeptide that do not affect the function of the polypeptide would be within the scope of the subject invention. Thus, the polypeptides disclosed herein should be understood to include variants and fragments, as discussed above, of the specifically exemplified sequences.

The subject invention further includes nucleotide sequences that encode the polypeptides disclosed herein. These nucleotide sequences can be readily constructed by those skilled in the art having the knowledge of the protein and amino acid sequences that are presented herein. As would be appreciated by one skilled in the art, the degeneracy of the genetic code enables the artisan to construct a variety of nucleotide sequences that encode a particular polypeptide or protein. The choice of a particular nucleotide sequence could depend, for example, upon the codon usage of a particular expression system or host cell.

Polypeptides having substitution of amino acids other than those specifically exemplified in the subject polypeptides are also contemplated within the scope of the present invention. For example, non-natural amino acids can be substituted for the amino acids of a polypeptide of the invention, so long as the polypeptide having substituted amino acids retains substantially the same activity as the polypeptide in which amino acids have not been substituted. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, ε-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs in general. Non-natural amino acids also include amino acids having derivatized side groups. Furthermore, any of the amino acids in the protein can be of the D- (dextrorotary) or the L- (levorotary) form.

Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby a polypeptide having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the polypeptide having the substitution retains substantially the same biological activity as a polypeptide that does not have the substitution. Table 1 provides a listing of examples of amino acids belonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

Also within the scope of the subject invention are polynucleotides that have the same, or substantially the same, nucleotide sequence of a polynucleotide exemplified herein, except for the presence of one or more nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, so long as these variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides exemplified herein (i.e., they encode a protein having the same amino acid sequence or the same functional activity as one of the polynucleotides specifically exemplified herein). Thus, the polynucleotides disclosed herein should also be understood to include variants and fragments thereof.

The methods of the present invention can be used with humans and other animals. The other animals contemplated within the scope of the invention include domesticated, agricultural, or zoo- or circus-maintained animals. Domesticated animals include, for example, dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs, monkeys or other primates, and gerbils. Agricultural animals include, for example, horses, mules, donkeys, burros, cattle, cows, pigs, sheep, and alligators. Zoo- or circus-maintained animals include, for example, lions, tigers, bears, camels, giraffes, hippopotamuses, and rhinoceroses. As used herein, the terms “patient” and “subject” are used interchangeably and are intended to include such human and non-human species. Likewise, in vitro methods of the present invention may also be performed on cells of one or more human or non-human, mammalian species.

As one of ordinary skill in the molecular biological arts can readily appreciate, there can be a number of variant sequences of a gene or polynucleotide found in nature, in addition to those variants that may be artificially prepared or synthesized by an ordinary-skilled artisan in a laboratory environment. The polynucleotides of the subject invention encompasses those specifically exemplified herein, as well as any natural variants thereof, as well as any variants which can be created artificially, so long as those variants retain the desired biological activity.

Also within the scope of the subject invention are polynucleotides which have the same nucleotide sequences of a polynucleotide exemplified herein except for nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, as long as these variant polynucleotides retain substantially the same relevant biological activity as the polynucleotides specifically exemplified herein. Thus, the polynucleotides disclosed herein should be understood to include variants and fragments, as discussed above, of the specifically exemplified sequences.

Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

The present invention also concerns pharmaceutical compositions comprising a vector system of the invention in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for topical or parenteral administration, comprising an amount of a compound constitute a preferred embodiment of the invention. The dose administered to a patient, particularly a human, in the context of the present invention should be sufficient to achieve a therapeutic response in the patient over a reasonable timeframe, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.

The subject invention also concerns kits comprising a vector system of the invention in one or more containers. Kits of the invention can optionally include pharmaceutically acceptable carriers and/or diluents. In one embodiment, a kit of the invention includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit of the invention includes instructions or packaging materials that describe how to administer a vector system contained within the kit to a selected mammalian recipient. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a vector system of the invention is provided in the kit as a solid. In another embodiment, a vector system of the invention is provided in the kit as a liquid or solution. In certain embodiments, the kits may include one or more ampoules or syringes that contain a vector system of the invention in a suitable liquid or solution form.

Multiple distinct AAV-based, dual-vector systems have been created and disclosed herein for use in gene-replacement therapies, including, for example, in the treatment of USH1B in human patients. In a specific embodiment, a vector system of the present invention employs two discrete AAV vectors that each packages a maximal-size DNA molecule (i.e., ˜4.5 to 4.8 Kb). The two vectors are co-administered to selected recipient cells to reconstitute the full-length, biologically-active, Myo7a polypeptide. In these constructs, a portion of overlapping nucleic acid sequence is common to each of the vector genomes (see FIG. 1). When co-delivered to suitable cells, the overlapping sequence region facilitates the proper concatamerization of the two partial gene cassettes. These gene cassettes then undergo homologous recombination to produce a full-length gene cassette within the cells (see FIG. 1). Shared components of exemplified embodiments of the dual-vector systems include the use of AAV inverted terminal repeats (TR), the small version of the chimeric CMV/chicken β-actin promoter (smCBA), human Myo7a (hMyo7a) cDNA sequence and the SV40 polyadenylation (pA) signal.

Exemplary Definitions

In accordance with the present invention, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like, include, but are not limited to, DNAs (including, but not limited to, genomic and/or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs, and/or tRNAs), nucleosides, as well as one or more nucleic acid segments obtained from natural sources, chemically synthesized, genetically modified, or otherwise prepared or synthesized in whole or in part by the hand of man.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and compositions are described herein. For purposes of the present invention, the following terms are defined below:

The term “effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.

The term “operably linked,” as used herein, refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.

The term “promoter,” as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription.

The term “regulatory element,” as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.

The term “substantially corresponds to,” “substantially homologous,” or “substantial identity,” as used herein, denote a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent sequence identity, and more preferably, at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More preferably still, highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to which it was compared.

The percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, in the case of sequence homology of two or more polynucleotide sequences, the reference sequence will typically comprise at least about 18-25 nucleotides, more typically at least about 26 to 35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or even 100 or so nucleotides.

When highly-homologous fragments are desired, the extent of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more of the sequence comparison algorithms well-known to those of ordinary skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988).

The term “subject,” as used herein, describes an organism, including mammals such as primates, to which treatment with the compositions according to the present invention can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, non-human primates such as apes; chimpanzees; monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.

The term “treatment” or any grammatical variation thereof (e.g., treat, treating, and treatment etc.), as used herein, includes but is not limited to, alleviating a symptom of a disease or condition; and/or reducing, suppressing, inhibiting, lessening, ameliorating or affecting the progression, severity, and/or scope of a disease or condition.

The term “vector,” as used herein, refers to a nucleic acid molecule (typically one containing DNA) that is capable of replication in a suitable host cell, or one to which another nucleic acid segment can be operatively linked so as to facilitate replication of the operably-nucleic acid segment. Exemplary vectors include, without limitation, plasmids, cosmids, viruses and the like.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 Dual-AAV Vector System 1: hMyo7a Coding Overlap

In the case of Dual-Vector System 1 (FIG. 2) the overlapping DNA sequence shared by both vector A and vector B consists of a 1350-bp coding region for the human Myo7a gene. This is the simplest system of the present invention, and appears to be highly efficient in terms of full-length gene reconstitution, and MYO7A expression. Advantageously, each vector is of standard AAV packaging size, and as such, each packages DNA with a high degree of efficiency, and is readily adaptable to conventional GMP standards. Such vectors are also readily characterized to permit requisite regulatory approval prior to use in humans.

Example 2 Dual-AAV Vector System 2: hMyo7a Intron 23 Splicing

In the case of Dual-Vector System 2 (FIG. 3) the overlapping DNA sequence is composed of the native intron 23 of human Myo7a. Vector A contains the coding sequence corresponding to the amino-terminal portion of the hMyo7a cDNA relative to intron 23 (hMyo7aNT) and the native splice-donor site, followed by the entire intron 23 of hMyo7a (minus the native acceptor site). Vector B contains the carboxy-terminal portion of the hMyo7a cDNA relative to intron 23 (hMyo7aCT), and the full intron 23 of Myo7a (minus the native splice-donor site), followed by the native splice-acceptor site. Upon co-delivery to suitable mammalian host cells, the DNA of vectors A and B recombine to form a reconstituted full-length gene cassette. The resulting RNA transcript will then ‘splice out’ the native intron. Alternatively, recombination and formation of the gene cassette can occur via the AAV TRs. In this case, the RNA transcript will ‘splice out’ the native intron23-TR-intron23 motif. In both cases, however, the resulting mRNA is that of the reconstituted full-length hMyo7a gene sequence.

Example 3 In Vitro Performance of System 1: hMyo7a Coding Overlap

HEK293 cells were infected simultaneously with vector A and vector B of Dual-Vector System 1 (FIG. 2) at a ratio of 10000:1 vg/cell for each vector. The AAV vectors were then packaged in AAV2 virions that contain three Y→F mutations in the capsid protein (see, for example, Zhong et al., 2008). As a positive control, cells were transfected with plasmid containing full-length hMyo7a under the control of smCBA. Protein was recovered from cells at 3-, 4-, 5-, 6- and 7-days' post-infection, and an antibody directed against MYO7A was used to assay for its presence in the infected cells via immunoblotting. The results are shown in FIG. 6A and FIG. 6B. The area inside the white box is magnified, and presented at higher contrast on the right. Starting at 3-days' post-infection, the full-length human MYO7a protein was visible; peak expression of the protein occurred around Day 5.

Example 4 In Vivo Performance of System 1: hMyo7a Coding Overlap

Six week old shaker-1 (Myo7a null) mice were sub-retinally co-injected with 1 μL of the same preparations of vector A and vector B of Dual-Vector System 1 used in the above in vitro study. Both vectors contained ˜1×10¹² vg/mL. Four weeks' post-injection, retinas from treated and untreated eyes were collected and immunohistochemistry (IHC) was performed using an antibody directed against MYO7A (see FIG. 7A and FIG. 7B). The area in green showed MYO7A-specific staining, while the areas in blue corresponded to the nuclear-specific, DAPI stain. In the treated eye, MYO7A expression was clearly visible, and it appeared to be restricted to photoreceptors—more precisely to the juncture of the photoreceptor inner and outer segments.

Example 5 AAV Dual Vectors Efficiently Deliver Oversized Genes

Animals. Shaker-1 mice carrying the 4626SB allele, an effective null mutation (Liu et al., 1999; Hasson et al., 1997), were used on the C57BL6 genetic background, and maintained and genotyped as described (Liu et al., 1999; Gibbs et al., 2003a). They were maintained on a 12-hr light/12-hr dark cycle, with exposure to 10-50 lux of fluorescent lighting during the light phase, and were treated according to federal and institutional animal care guidelines. Homozygous mutants were distinguished from the heterozygous controls by their hyperactivity, head-tossing and circling behavior (Gibson et al., 1995), and/or by a PCR/restriction digest assay.

Construction of AAV Vectors. Single-vector platform: AAV vector plasmid, containing the truncated chimeric CMV/chicken β-actin promoter (smCBA) (Haire et al., 2006) and MYO7A cDNA was constructed by removing the full MYO7A cDNA from pEGFP-C2 by EagI and SalI digest, and then ligating into pTR-smCBA-GFP that had been digested with NotI and SalI to remove GFP. The MYO7A cDNA (˜6.7 kb) corresponded to isoform 2 of human MYO7A, and was the same as that described previously by Hashimoto et al. (2007), which was based on the sequence published by Chen et al. (1996) (see, SEQ ID NO:8). MYO7A isoform 2 is 114-kb shorter than isoform 1 (Chen et al., 1996; Weil et al., 1996). Both the MYO7A cDNA, and the resulting junctions were fully sequenced prior to packaging. All vectors intended for in vitro analyses were separately packaged in wild type AAV2, or alternatively in the AAV2(tripleY→F) capsid mutant vector (Petrs-Silva et al., 2011). As noted above, AAV2-based vectors were chosen for the in vitro experiments due to their increased transduction efficiency relative to other serotypes (Ryals et al., 2011). All vectors were packaged, purified, and titered using standard methods as previously described (Zolotukhin et al., 2002; Jacobson et al., 2006). Human embryonic kidney (HEK293) cells were transfected by the calcium phosphate method with vector plasmid carrying the full-length MYO7A coding sequence of variant 2 (the plasmid used to package fAAV). These transfected cells were then used as a positive control throughout immunoblot analyses to indicate the appropriate size of full-length MYO7A protein. Vector infections were carried out in HEK293 cells with titer-matched AAV vectors. In brief, cells were grown to 60-70% confluency. All vectors were diluted in a balanced salt solution to achieve the desired multiplicity of infection (MOI). If not specifically mentioned, cells were infected at 10,000 genome-containing particles/cell of each vector, resulting in an MOI of 20,000 total for each vector pair. Cells were incubated in medium containing 10% serum for 3 days post-infection at 37° C. under 7% CO₂, and then analyzed via immunoblot. Titers of 10¹² to 10¹³ particles/mL were obtained for different lots of AAV2-MYO7A and AAV5-MYO7A.

Oligonucleotide Sequences. For in vivo studies, a human influenza hemagglutinin (HA) tag was added to the 3′ termini of the full-length, simple overlap, trans-splicing, and hybrid 3′ vectors by utilizing a unique BamHI site (P19), and replacing the non-tagged 3′-end with an HA-tagged (P20) version. All constructs were sequence verified by Sanger sequencing.

AAV Vector Plasmid Design and Cloning. The full-length coding sequence of MYO7A (human isoform 2; GenBank Accession No. NM_001127180) was cloned into a vector plasmid containing the strong, ubiquitous CMV/chicken β-actin (smCBA) promoter (Haire et al., 2006), a polyadenylation signal, and the AAV2 ITRs. Packaging of this plasmid generated the fAAV vector (FIG. 22A). In all systems, the 5′ vectors shared the smCBA promoter and a 5′ portion of MYO7A, whereas the 3′ vectors contained a 3′-portion of MYO7A, and a bovine growth hormone (bGH) polyadenylation signal. Oligonucleotides used for vector construction are listed in Table 2. The simple overlap contained nucleotides 1 through 3644 of MYO7A cDNA from the ATG in the 5′ vector, and nucleotides 2279 through 6534 in the 3′ vector. The fragments were amplified with oligonucleotides P1 and P3 by polymerase chain reaction (PCR) and cloned into the 5′ vector via NotI and NheI, and the 3′ vector with P3 (AflII) and P4 (KpnI), respectively. The resulting two vector plasmids share 1365 bp of overlapping MYO7A sequence (FIG. 22B). The trans-splicing and hybrid vectors utilize splice junctions composed of either ideal splice donor and acceptor sites derived from AP coding sequence or native MYO7A splice junctions from exons 23 and 24 (Yan et al., 2002). To create the 5′ trans-splicing vector, the splice-acceptor site was amplified using oligonucleotides P5 and P6 (NheI), and the amplicon was then used in a second reaction with oligonucleotide P7 (NsiI) to add a part of the MYO7A coding sequence for cloning. The corresponding 3′ vector was similarly created by amplifying the splice-acceptor site with oligonucleotides P8 (AflII) and P9 in a first PCR, and adding part of the 3′ MYO7A coding sequence with oligonucleotide P10 (AgeI) in a second PCR (see FIG. 22C). The AP hybrid vectors were created by adding 270 bp of AP overlap sequence to the respective trans-splicing vectors (Ghosh et al., 2011). The sequence was amplified by PCR and, in so doing, appropriate restriction endonuclease sites were added. For the 5′ vector oligonucleotides P11 (NheI) and P12 (SalI) were used, while oligonucleotides P13 (Nod) and P14 (AflII) were used for the 3′ vector (FIG. 22D). A fourth vector pair, “native intron hybrid” vector, was also created to exploit the natural sequence in and around intron 23 of MYO7A as a recombination locus, and subsequent splicing signal. The 5′-portion was created by amplifying intron 23 with oligonucleotides P15 and P16 (NheI) first, and then using the resulting amplicon in a second reaction with oligonucleotide P7 (NsiI) to facilitate cloning. The corresponding 3′-vector was constructed by amplifying the intron 23 with oligonucleotides P17 and P18 (AflII), and the resulting amplicon, with oligonucleotide P10 (AgeI) in a second reaction (see FIG. 22E).

TABLE 2 OLIGONUCLEOTIDES USED IN THIS STUDY Restriction OliGo 5′-3′ sequence (restriction sites underlined) site (SEQ ID NO:) P1 GCGGCGGCCGCCACCATGGTGATTCTTCAGCAGGGGGAC NotI (SEQ ID NO: 9) P2 GCGGCTAGCGAAGTTCCGCAGGTACTTGAC NheI (SEQ ID NO: 10) P3 GCGCTTAAGCAGGTCTAACTTTCTGAAGCTG AflII (SEQ ID NO: 11) P4 GCGGGTACCTCACTTGCCGCTCCTGGAGCC KpnI (SEQ ID NO: 12) P5 GGCACCTAGTGGCTTTGAGGTAAGTATCAAGGTTACAAGAC (SEQ ID NO: 13) P6 GCGGCTAGCTCAGAAACGCAAGAGTCTTC NheI (SEQ ID NO: 14) P7 CTTCTTTGTGCGATGCATCAAG Nsii (SEQ ID NO: 15) P8 GCGCTTAAGCGACGCATGCTCGCGATAG AflII (SEQ ID NO: 16) P9 CGCCCTCGCTCCAGGTCCTGTGGAGAGAAAGGCAAAG (SEQ ID NO: 17) P10 GAACCCGAACCGGTCCTTG AgeI (SEQ ID NO: 18) P11 GCGGCTAGCCCCCGGGTGCGCGGC NheI (SEQ ID NO: 19) P12 GCGGTCGACGAAACGGTCCAGGCTATGTG SalI (SEQ ID NO: 20) P13 GCGGCGGCCGCCCCCGGGTGCGCGGCG NotI (SEQ ID NO: 21) P14 GCGCTTAAGGAAACGGTCCAGGCTATGTG AflII (SEQ ID NO: 22) P15 CAGGCACCTAGTGGCTTTGAGGTACCAGGCTAGGGACAGG (SEQ ID NO: 23) P16 GCGGCTAGCCGCCTGAGCCCAGAAGTTC NheI (SEQ ID NO: 24) P17 CGCCCTCGCTCCAGGTCCTGAAGGAGACAAGAGGTATG (SEQ ID NO: 25) P18 GCGCTTAAGCACCGCTTGTGTTGATCCTC AflII (SEQ ID NO: 26) P19 GCCAGGGAAGGATCCCATG BamHI (SEQ ID NO: 27) P20 GCGGGTACC TCATGCGTAATCCGGTACATCGTAAGGGTACTTGCCGCTCCT KpnI (SEQ ID NO: 28) GGAGCC P21 AGCTTCGTAGAGTTTGTGGAGCGG (SEQ ID NO: 29) P22 GAGGGGCAAACAACAGATG (SEQ ID NO: 30) Oligonucleotides were used to make 5′ and 3′ vectors of the dual-vector platforms (P1-P20). Oligonucleotides were used to characterize the fidelity of the overlap in simple overlap, trans-splicing and AP hybrid vector platforms (P21-P22). Restriction sites used for cloning are underlined and the introduced HA tag is noted in italics (P19).

Dual-Vector Platform. Two separate vector plasmids were constructed: Vector A contains the strong, ubiquitous “smCBA” promoter and MYO7A cDNA encoding the N-terminal portion. Vector B contains MYO7A cDNA encoding the C-terminal portion and a poly-A signal sequence. Each vector plasmid contained both inverted terminal repeats (ITRs). Using PCR with full-length MYO7A cDNA as a template, the MYO7A cDNA was divided roughly in half with amplicons encompassing nucleotide positions 1 through 3644 (Vector A) and 2279 through 6647 (Vector B) relative to ATG start position 1. The resulting two-vector plasmids shared 1365 bp of overlapping MYO7A sequence, and were 5.0- and 4.9-Kb in length, respectively. This was well within the size limitation of standard AAV vectors. Both vector plasmids were sequence verified and separately packaged by standard AAV production methods (Zolotukhin et al., 2002; Jacobson et al., 2006). The titer of the first lot contained 2.5×10¹² particles/mL of each vector, and the second lot contained 4×10¹² particles/mL of each vector.

Reverse Transcription and Characterization of Overlap Region. HEK293 cells were infected with dual vectors, and total RNA was extracted with the RNeasy® kit (Qiagen, Hilden, Germany) according to the manufacturer's recommended protocol. Two micrograms of RNA was then subjected to DNaseI (NEB) digestion for 30 min at 37° C., followed by heat inactivation at 75° C. for 10 min. Reverse transcription to cDNA was achieved with the SuperscriptIII® kit (Life Technologies, Grand Island, N.Y., USA) according to the standard protocol utilizing the oligo dT primer. Two microliters of cDNA was used as template in a PCR (95° C. for 3 min initial denature, 35 cycles of 95° C. for 45 sec, 55° C. for 45 sec, 72° C. for 12 min, and a final 72° C. for 15 min) using oligonucleotide primers P21 and P22 (see Table 2). Annealing sites for these primers are located 5′ and 3′, respectively, of the area of cDNA overlap (in other words, outside the region of overlap) in the simple overlap and hybrid vector pairs. The 3′-primer annealed to sequence that was complimentary to the bGH polyA. Resulting products were digested with either PpuMI or BglII, separated on a 1.5% agarose gel, and subsequently analyzed on a UV screen. Separately, products were digested with KpnI and AgeI, and subsequently cloned into a pUC vector for sequencing of the entire overlap region. M13 forward and reverse primers that were specific for the vector were used to obtain sense and antisense reads resulting in an ˜140 bp overlap of the sense and antisense reads. To demonstrate that these methods were capable of detecting aberrant sequence (i.e., quality control), a MYO7A sequence was generated using either an artificial insertion (HindIII fill-in at position 2635) or a point mutation (T→C) at position 2381, and the the analyses were repeated.

Viral Delivery in Vitro. HEK293A cells (Invitrogen), grown in DMEM with 10% FBS and 1×NEAA and Pen/Strep (Invitrogen) were plated in 6 well-plates. The next day cells were incubated, at 37° C. and 5% CO₂, with AAV2- and AAV5-MYO7A at an MOI of 10,000 viral particles/cell in 500 μL of complete medium, containing also 40 μM of calpain inhibitor (Roche, Pleasanton, Calif., USA). Two hours later complete medium was added. The next day, the medium was changed and cells were incubated for an additional 48 hrs. Alternatively, some cells were transfected with 1 μg of vector pTR-smCBA-MYO7A, complexed with Lipofectamine 2000 (ratio 1:3), according to the manufacturer's instructions (Invitrogen).

Primary mouse RPE cells were derived from P14-P16 Myo7a-null animals and cultured in 24-well dishes, as described (Gibbs et al., 2003a; Gibbs and Williams, 2003b). After 48 hrs in culture, cells were transduced with viruses. Cells were incubated in 100 μL of complete medium containing 40 μM of calpain inhibitor, and 10,000 viral particles/cell from full-strength AAV stocks. After 2 hrs, 400 μL of complete medium was added to each well, and incubated overnight. The medium was changed the following day, and cells were incubated for an additional 48 hrs.

ARPE19 cells (American Type Culture Collection, Manassas, Va., USA) were cultivated in DMEM/F-12 with 10% FBS and split into 24-well plates with glass coverslips. Cells were grown to confluency and then transduced in the same manner, as were the primary RPE cells.

MYO7A expression analysis by Western blot and Immunofluorescence. HEK293A and primary mouse RPE cells that were transduced with AAV-MYO7A were collected 3 days post-transduction. For western blot analyses, cells were collected and lysed in 20 mM TRIS, pH 7.4, 5 mM MgCl₂, 10 mM NaCl, 1 mM DTT and 1× protease inhibitor cocktail (Sigma-Aldrich Chemical Co., St. Louis, Mo., USA). Equivalent amounts of total protein were separated on a 7.5% SDS-PAGE gel. After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa City, Iowa USA) (Soni et al., 2005), and mouse anti-actin antibody (Sigma-Aldrich) as a loading control.

Immunofluorescence was performed with ARPE19 and mouse RPE primary cells, 3 days after infection. Cells were fixed in 4% formaldehyde, blocked with blocking solution (0.5% BSA/0.05% saponin in PBS), incubated with the mouse anti-MYO7A followed by goat anti-mouse Alexa-568 (Molecular Probes, Carlsbad, Calif., USA). Coverslips were mounted with mounting medium containing DAPI (Fluorogel II, Electron Microscopy Sciences, Hatfield, Pa., USA) and visualized on a Leica confocal system.

Protein extraction and immunoblotting. Transfected and infected HEK293 cells were harvested and washed twice in PBS and processed as previously reported with minor modifications (Boye et al., 2012). The cells were lysed by 3×30 sec pulses of sonication in 200 μL of sucrose buffer (0.23 M sucrose, 2 mM EDTA, 5 mM Tris-HCl, pH 7.5) containing protease inhibitors (Roche, Mannheim, Germany). Unlysed cells and cell debris were removed by centrifugation at 14,000 rpm for 10 min. The protein concentration of the supernatant was measured with BCA (Thermo Fisher Scientific, Rockland, Ill., USA). Equal amounts of protein were then loaded on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels (BioRad, Hercules, Calif., USA) and transferred in CAPS buffer (pH 11) onto PVDF membranes (Millipore, Billerica, Mass.). Blots were then labeled with antibodies against MYO7A (monoclonal antibody raised against amino acids 11-70 of human MYO7A; Santa Cruz, Dallas, Tex., USA; 1:1000) or HA (MMS-101P; Covance, Gaithersburg, Md., USA; 1:500) and β-actin (ab 34731; Abcam, Cambridge, Mass., USA; 1:5000). For visualization with the Odyssey system (Li-Cor, Lincoln, Nebr., USA), an antimouse and an anti-rabbit secondary antibody conjugated with CW800 and IR680 dyes (Li-Cor), respectively, were used. Semiquatitative densitometric measurements were performed with Odyssey acquisition and analysis software (Li-Cor). The dual-color images were separated in their respective channels and converted to gray scale for presentation purposes. Size markers present in one channel of each blot were added to both channels for visualization of protein sizes.

Viral Delivery in vivo. Mice were anesthetized with 2.0-3.0% isoflurane inhalation. The pupils of the animals were dilated with 1% (wt./vol.) atropine sulfate and 2.5% phenylephrine. A local anesthetic (0.5% proparacaine hydrochloride) was also administered. A sclerotomy in the temporal limbus was performed with a 27-Ga needle. A 32-Ga blunt needle, attached to a microsyringe pump (WPI, Sarasota Fla., USA) was inserted and 1 μL of viral solution was injected into the ventral subretinal space of P14-P16 animals. Retinal detachment was visualized under a dissecting microscope, and registered as indication of a positive subretinal injection. One microliter of the following AAV8(Y733F)-based vectors was injected subretinally in one eye of C57BL/6 mice: single fAAV (1×10¹³ vg/mL), front and back half “hybrid” vectors combined equally (each vector=1×10¹³ vg/mL), or front and back half “simple overlap” vectors combined equally (each vector=1×10¹³ vg/mL). Subretinal injections were performed as previously described (Timmers et al., 2001). Further analysis was carried out only on animals that received comparable, successful injections (>60% retinal detachment with minimal surgical complications).

Light Microscopy and Immunoelectron Microscopy of Retinas. Eyecups were processed for embedment in either LR White or Epon, and semithin and ultrathin sections were prepared. Semithin sections were stained with toluidine blue and visualized on a Leica confocal system. Ultrathin sections were labeled with purified MYO7A pAb 2.2 (Liu et al., 1997) and monoclonal anti-opsin (1D4, R. Molday), followed by gold-conjugated secondary antibodies (Electron Microscopy Sciences), as described previously (Lopes et al., 2011). Negative control sections processed at the same time included those from Myo7a-null retinas, and, as positive control, WT animals were used.

MYO7A immunogold density was determined on sections of age-matched WT, Myo7a-null retinas and retinas of Myo7a-null animals that had been injected with AAV-MYO7A at P14-16 and dissected three weeks later. For quantification of the immunolabel, all of the gold particles in a complete section of each RPE cell were counted. The area of each cell's profile was determined using ImageJ software. For background labeling, the concentration of label in sections of untreated Myo7a-null animals was measured. Data were expressed with this background labeling subtracted.

The concentration of MYO7A and opsin immunogold labeling in the connecting cilia of photoreceptor cells was determined by counting gold particles along longitudinal profiles of connecting cilia and measuring the length of each profile.

Analysis and quantifications were performed in a minimum of three different retinas, from three different animals. Statistical analysis was performed using one-tail Student's t-test.

Six weeks postinjection, C57BL/6 mice were enucleated and their eyes processed and immunostained as previously described (Boye et al., 2011) with minor modifications. Retinas were immunostained with an antibody specific for HA (monoclonal Ab clone 12CA5; Roche), counterstained with DAPI, and imaged with a spinning disk confocal microscope (Nikon Eclipse TE2000 microscope equipped with Perkin Elmer Ultraview Modular Laser System and Hamamatsu O-RCA-R2 camera). Images were obtained sequentially using a 20× (air) objective lens. All settings (exposure, gain, laser power) were identical across images. All image analysis was performed using Volocity 5.5 software (Perkin Elmer, Waltham, Ma., USA).

Results

AAV-MYO7A single vector preparations. AAV vector plasmid was engineered to contain a truncated chimeric CMV/chicken β-actin promoter, smCBA (Haire et al., 2006) and the 6.7-kb cDNA encoding the full-length isoform 2 of human MYO7A (NCBI #NM_001127180) (FIG. 10A). The smCBA promoter exhibits the same tropism and activity in mouse retinas as that of the full-length CBA promoter (Haire et al., 2006; Pang et al., 2008). Titers of 10¹² to 10¹³ particles/mL were obtained for different lots of AAV2-MYO7A and AAV5-MYO7A. A concentration of 10¹² particles/mL was regarded as the standard concentration (1×), from which dilutions were made. The experiments were performed with virus obtained from three separate preparations. No differences in expression or phenotype correction, as described below, were observed among the different lots for AAV2-MYO7A or AAV5-MYO7A at a given concentration.

MYO7A Expression in Cell Culture. Transduction of primary cultures of Myo7a-null RPE cells with 1× single AAV2-MYO7A or AAV5-MYO7A resulted in the expression of a polypeptide that, by western blot analysis, had an apparent mass that was comparable to that of WT MYO7A protein, and was present at similar levels to that found in primary cultures of Myo7a^(−/−) RPE cells (FIG. 10B). Likewise, a single band of appropriate size was detected on western blots of HEK293A cells. Immunofluorescence of the primary RPE cells showed that the MYO7A protein, resulting from 1× single AAV-MYO7A treatment of MYO7A-null cells, had a subcellular localization pattern that was comparable to that of endogenous MYO7A in control cells, indicating the generation of appropriately targeted protein (FIG. 10C, FIG. 10D, FIG. 10E, and FIG. 10F). ARPE19 cells were also infected with 1× or diluted (1:100) AAV2-MYO7A or AAV5-MYO7A, and compared with non-treated cells. An increase in MYO7A immunofluorescence was detected in the treated cells, and the intracellular localization of the label was comparable to that in untreated cells (FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F).

Localization of MYO7A in Vivo. Most retinal MYO7A is found in the RPE (Hasson et al., 1995), however, the protein is also present in the connecting cilium and pericilium of the photoreceptor cells (Liu et al., 1997; Williams, 2008). A diagram illustrating this distribution and the retinal functions of MYO7A has been published in a recent review (Williams and Lopes, 2011).

Three weeks following injection of 1×AAV2-MYO7A or AAV5-MYO7A into the subretinal space of Myo7a-null mice, retinal tissue was examined by immunoelectron microscopy to test for MYO7A expression. Immunogold label was evident in the photoreceptor cells, where it was localized in the connecting cilium and pericilium, comparable to that in WT retinas (FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E). Label was also present throughout the RPE cells, particularly in the apical cell body region (FIG. 11F, FIG. 11F-1, FIG. 11G, and FIG. 11G-1; see FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D for controls), as found in WT retinas (Gibbs et al., 2004; Liu et al., 1997).

MYO7A has a similar distribution in both rod and cone photoreceptor cells (Liu et al., 1999). To test whether treatment with AAV-MYO7A also affected cone photoreceptor cells, it was determined whether MYO7A was also present in the ciliary region of cone photoreceptors. Double immunoEM of treated retinas was performed, using a MYO7A antibody together with an antibody specific for rod opsin. Although there are only a small number of cones with aligned connecting cilia found in each ultrathin section, MYO7A immunogold label was evident in the connecting cilium and periciliary region of these cones, which were identified by lack of rod opsin labeling in their outer segments (in contrast to the surrounding rod outer segments) (FIG. 11H and FIG. 11I). Hence, AAV2-MYO7A and AAV5-MYO7A can transduce cone as well as rod photoreceptor cells.

Dose-dependent MYO7A expression in photoreceptor and RPE cells. To determine the levels of MYO7A expression following treatment with different concentrations of AAV2-MYO7A and AAV5-MYO7A (1×, 1:10 or 1:100 dilutions), MYO7A immunogold labeling was quantified in EM images, taken within 1.4 mm of the injection site. Reliable detection of MYO7A in the photoreceptor cells, where its distribution is limited to the connecting cilium and pericilium, requires the higher resolution provided by electron microscopy (Liu et al., 1997). Immunogold particle density was measured in images of the photoreceptor connecting cilium and pericilium, shown in complete longitudinal section (from the basal bodies to the base of the outer segment), and in images showing the RPE cells in apical to basal section. Particle density was expressed as particles per length of cilium for the photoreceptor cells (each connecting cilium is ˜1.2 μm long), and as particles per area for the RPE cells (the entire area between the apical and basal surfaces was included). Particle density is dependent on exposure of epitopes on the surface of the section, and, as such, provides a relative linear measure of antigen density under the conditions used here (i.e., grids were etched and labeled in an identical manner, and the labeling was not so dense as to be affected by steric hindrance).

Treatment with 1×AAV2-MYO7A or AAV5-MYO7A resulted in 2.5-2.7 times the density of immunolabel in the photoreceptor cilium, compared with that found in WT retinas, while the 1:10 and 1:100 dilutions resulted in a density of immunolabel that was more comparable to WT levels (FIG. 11J, FIG. 11L, and FIG. 19). Quantification of immunogold label in the RPE showed that injection of AAV2-MYO7A resulted in 2.7 times more label than in WT, with the 1:10 and 1:100 dilutions showing no significant difference (FIG. 11K). In contrast, the level of MYO7A immunolabel in the RPE of retinas injected with AAV5-MYO7A varied in relation to virus titer, with the full dose virus effecting 2.2-fold more MYO7A than that found in WT RPE, the 1:10 dilution effecting WT levels, and the 1:100 dilution resulting, on average, ˜60% of WT levels (FIG. 11M).

These counts of labeling density indicate that 1×AAV-MYO7A resulted in more than double the normal level of MYO7A expression in both the photoreceptor and RPE cells. The distribution of MYO7A was not affected by this overexpression in the photoreceptor cells. In the RPE cells, the overall distribution of MYO7A was comparable to WT, with a higher concentration in the apical cell body region. However, with 1×AAV2-MYO7A or 1×AAV5-MYO7A, the proportion of MYO7A that was associated with melanosomes was only 55% of that in WT RPE: This difference is possibly because the proteins that link MYO7A to the melanosomes, MYRIP and RAB27A (Klomp et al., 2007; Lopes et al., 2007), may have remained near WT levels, and thus limited the absolute amount of MYO7A that could associate with the melanosomes.

Despite the overexpression of MYO7A, no pathology was evident in retinas, up to 3 months after injection of 1× (or 1:10) AAV2-MYO7A. However, two out of six retinas injected with 10¹³ particles/mL of AAV5-MYO7A (i.e., 10×) showed evidence of photoreceptor cell loss across the retina after 3 weeks (AAV2-MYO7A was not tested at this titer) (FIG. 19).

Correction of melanosome localization in the RPE. In Myo7a-mutant mice, melanosomes are absent from the apical processes of the RPE cells (Liu et al., 1998). This mutant phenotype is evident at all neonatal ages, and is due to loss of actin-based transport of the melanosomes by the myosin 7a motor (Gibbs et al., 2004). Three weeks following injection of 1×AAV2-MYO7A or AAV5-MYO7A into the subretinal space of Myo7a-null mice, melanosomes were observed to have a normal distribution in all RPE cells near the site of injection (within 1.4 mm) (n=10 each for AAV2-MYO7A and AAV5-MYO7A) (FIG. 12A, FIG. 12B, and FIG. 12C). Well away from the injection site, a mixture of corrected and uncorrected RPE cells was evident, while, at the periphery of the retina, the cells all exhibited the Myo7a-mutant phenotype, indicating lack of correction in this region (FIG. 12D, FIG. 12E, and FIG. 12F). The correction of melanosomes was still evident in retinas that were fixed 3 months after injection (FIG. 20). Correction was also observed in all eyes injected with 1:10 dilution AAV2-MYO7A (n=6) or AAV5-MYO7A (n=6), as well as in all eyes injected with 1:100 dilution AAV2-MYO7A (n=6) or AAV5-MYO7A (n=6), although with the 1:100 dilution some of the RPE cells near the site of injection were not corrected.

Correction of opsin distribution. Myo7a-mutant mice have an abnormal accumulation of opsin in the connecting cilia of the photoreceptor cells, a phenotype that is evident by immunoEM with opsin antibodies (Liu et al., 1999). This mutant phenotype suggested that myosin 7a functions in the vectorial delivery of opsin to the outer segment (Liu et al., 1999). Quantification of immunogold opsin labeling in the connecting cilia, demonstrated that this phenotype was corrected with 1×AAV2-MYO7A or AAV5-MYO7A (FIG. 13; FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D). This analysis also showed phenotype correction with 1:100 dilutions, although the data indicated that a full WT phenotype was not achieved (FIG. 13), despite WT levels of MYO7A (FIG. 11J and FIG. 11L), suggesting that some of the MYO7A may not be fully functional.

AAV2-MYO7A dual-vector preparations. The preceding results demonstrate that a single AAV vector is capable of delivering functional MYO7A to the RPE and photoreceptor cells in vivo. Because the size of smCBA-MYO7A is ˜2 kb larger than the nominal carrying capacity of an AAV (Grieger and Samulski, 2005), this transduction may involve undefined fragmentation of the smCBA-MYO7A cDNA followed by reassembly of plus and minus cDNA strands after delivery to the cell as shown for other large genes (Dong et al., 2010; Lai et al., 2010; Wu et al., 2010). To evaluate whether two AAV vectors containing defined, overlapping fragments of MYO7A cDNA (1365 bases) were also capable of mediating full-length MYO7A expression, an AAV2-based dual-vector system (FIG. 14A-1 and FIG. 14A-2) was developed. Two separate lots of the AAV2-MYO7A(dual vector) were prepared, each containing equal concentrations of AAV2-smCBA-MYO7A(5′-half) and AAV2-MYO7A(3′-half). The titer of the first lot contained 2.5×10¹² particles/mL of each vector, and the second lot contained 4×10¹² particles/mL.

MYO7A expression with AAV2 dual vectors. Western blot analysis of primary cultures of Myo7a-null RPE cells, infected with AAV2-MYO7A(dual vector) of either lot, showed that the cells expressed a MYO7A-immunolabeled polypeptide of comparable mass to that of WT MYO7A (FIG. 14B). However, the expression level of MYO7A in the Myo7a-null RPE cells was significantly less than that found in primary cultures of Myo7a^(+/−) RPE cells (cf. lanes 2 and 3 in FIG. 14B), unlike that found for the single AAV2 or AAV5 vectors (FIG. 10B). Quantitative analysis of western blots showed that Myo7a-null RPE cells, transduced with the single vectors (1×), AAV2-MYO7A or AAV5-MYO7A, or with AAV2-MYO7A(dual vector), expressed MYO7A at levels that were 82%, 111%, and 10%, respectively, of the level of MYO7A in Myo7a^(+/−) RPE cells.

FIG. 16 is a Western blot using the same dual-vector system as above except in an AAV8 serotype. FIG. 16 shows expression level of myo7A using the dual-vector system that was nearly equivalent to the wild type myo7A expression level. While the reasons for the discrepancy are unclear, much better results were obtained by the inventors using the dual-vector systems than were obtained by several outside collaborators. Wild type like levels of MYO7A expression was observed in shaker-1 retinas following injection with dual-AAV8(Y733F) vectors. Thus, very good expression of MYO7A with the dual AAV platform has been achieved.

Immunofluorescence of primary Myo7a-null RPE cells, infected with AAV2-MYO7A(dual vector), showed that a few cells scattered throughout the culture exhibited very high levels of MYO7A, but all other cells contained insignificant levels (FIG. 14C, FIG. 14D, and FIG. 14E). The cells overexpressing MYO7A typically had altered morphology, suggesting that the high levels of MYO7A may be toxic. Similarly, immunofluorescence of ARPE19 cells, infected with AAV2-MYO7A(dual vector), resulted in a minority of cells that were labeled intensely with MYO7A antibody, with most of the cells appearing to express only endogenous levels of MYO7A (FIG. 14F and FIG. 17F).

Immunolabeling of retinas, prepared 3 weeks after subretinal injection with AAV2-MYO7A(dual vector) of either lot, also showed only a few RPE cells and photoreceptor cells with clear MYO7A expression, although significant overexpression was not evident in this in vivo experiment. Immunogold particle counts from images of ultrathin sections were used to quantify the level of MYO7A expression in Myo7a-null retinas that were treated with the second lot of AAV2-MYO7A(dual vector). Within 1.4 mm of the injection site, MYO7A immunolabeling of the connecting cilium and pericilium of the photoreceptor cells was a mean of 48% of that in WT retinas: 2.8 particles/um (n=3 retinas) compared with 6.5 particles/μm for WT (n=3 retinas). The mean label density in apical-basal sections of the RPE was 35% of that in WT retinas: 11 particles/100 μm² compared with 31 particles/100 μm² for WT. However, it was clear that these lower means were achieved by some cells expressing near normal amounts of MYO7A and the majority expressing very little; over half the cells had fewer than 10 particles/100 μm² (FIG. 14G).

Correction of Myo7a-mutant phenotypes with AAV2 dual vectors. Eyes were analyzed for correction of melanosome localization and ciliary opsin distribution within 1.4 mm of the injection site. With either lot of AAV2-MYO7A(dual vector), some RPE cells (29% for lot 1 treatment [n=6 retinas], 35% for lot 2 treatment [n=9 retinas]) were observed to have a normal apical melanosome distribution, but most of the cells in this region retained the Myo7a-mutant phenotype, resulting in a mosaic effect (FIG. 15A) that contained a much lower proportion of corrected cells than that observed with a 1:100 dilution of either of the single vectors. The only correction observed in 3 eyes injected with a 1:10 dilution of AAV2-MYO7A(dual vector) (first lot), was in 18% of the RPE cells in one of the retinas. With full-strength of AAV2-MYO7A(dual vector) (second lot), opsin immunogold density averaged 3.2±0.4 particles/μm of cilium length, which was reduced from untreated retinas (4.2±0.8 particles/μm; p=0.003), but still greater than WT levels (1.1±0.2 particles/μm), suggesting that most cells were not corrected.

Using immunoelectron microscopy, a correlation between phenotype correction and the expression level of MYO7A was identified (determined by the mean concentration of immunogold particles in an apical-basal section of each RPE cell) (FIG. 15B, FIG. 15C, FIG. 15D, and FIG. 15E). From the eyes injected with AAV2-MYO7A(dual vector) (second lot), it was shown that the corrected RPE cells contained a mean of 108% of the WT level of MYO7A (the minimum level was 82%). RPE cells that were not corrected contained a mean of 26% of the WT level of MYO7A (the maximum level was 92%). While these data showed that higher expression of MYO7A is correlated with phenotype correction (FIG. 15F), it also indicated that some of the labeled MYO7A protein was not functional, given that melanosomes are localized normally in mice that are heterozygous for the Myo7a-null allele and have only ˜50% of the WT level of MYO7A.

Expression of MYO7A with simple overlap vectors. AAV2-based simple overlap vectors were evaluated in vitro at a variety of MOIs to evaluate how the concentration of vector pairs related to MYO7A expression. How levels of MYO7A changed over time was also evaluated in infected cells. HEK293 cells were infected with simple overlap vector pairs packaged in AAV2(tripleY-F) vector (FIG. 23A). A preliminary co-infection with AAV2(tripleY-F) simple overlap vectors (MOI of 10,000 for each vector) indicated that MYO7A is expressed, and that migration of the protein on gel is identical to a full-length transfection control (FIG. 23A). Coinfection at MOIs of 400, 2000, and 10,000 of each vector shows that the efficiency of the simple overlap system is proportional to the amount of 5′ and 3′ vectors used (FIG. 23B). MYO7A expression increased as a function of incubation time up to 5 days postinjection in HEK293 cells (FIG. 23C). The visible expression decline was because of a reduction of viable cells in the culture vessel at the later times.

Comparison of fAAV-MYO7A to dual-AAV-MYO7A expression and evaluation of AAV serotype efficiency. Previously, it was shown that fAAV-MYO7A was able to ameliorate the retinal phenotype of the shaker1 mouse (Colella et al., 2013; Lopes et al., 2013; Trapani et al., 2013). To provide a basis for comparison dual-AAV-vector expression was evaluated relative to fAAV in vitro. After infection in HEK293 cells, all dual-vector systems expressed MYO7A more efficiently than fAAV (FIG. 24). The AP hybrid platform showed the strongest expression, followed by the simple overlap system.

Other studies have shown, in the context of a conventionally sized DNA payload, that the transduction efficiency and kinetics of AAV2(tripleY-F) vectors are increased relative to standard AAV2 both in vitro and in vivo (Li et al., 2010; Markusic et al., 2010; Ryals et al., 2011). The efficiency of AAV2 versus AAV2(tripleY-F) dual vectors was directly compared in HEK293 cells. Surprisingly, standard AAV2-mediated MYO7A expression was higher than that seen with titer-matched AAV2(tripleY-F) (FIG. 24). Identical results were obtained when comparing different AAV2 and AAV2(tripleY-F) dual-vector preparation packaged with identical vector plasmid.

Comparison of relative efficiencies and specificity of full-length MYO7A expression. To quantitatively evaluate the relative expression efficiencies of the dual-vector platforms and to assess specificity of full-length protein, HEK293 cells were infected with either the 5′ and 3′ AAV2-based vector pairs combined or the corresponding 5′ vector alone. An additional hybrid vector pair was included that incorporated native MYO7A intronic sequence (intron 23) that served as overlapping sequence and provided appropriate splicing signals. All 5′ vectors produced low amounts of a defined, less than full-length peptide detectable on Western blot with the exception of the simple overlap vector (FIG. 25A). However, the trans-splicing and the AP hybrid platforms revealed a distinct decrease of this undesired product when the 3′ vector was added to the sample (FIG. 25A). The native intron hybrid platform also showed this secondary band on Western blots, again suggestive of a truncated protein originating from the 5′ vector alone. In contrast to all other platforms tested, this band intensity increased with the addition of the 3′ vector. Each platform's relative ability to promote reconstitution was compared by quantifying the amount of 5′ vector-mediated truncated protein product in the presence or absence of the respective 3′ vector (FIG. 25B). Full-length MYO7A expression on Western blot was then quantified relative to transfection control (FIG. 25C). AP hybrid-mediated MYO7A was the strongest followed by simple overlap, trans-splicing, and native intron hybrid (FIG. 25C).

Characterization of the overlap/splice region of the expressed MYO7A. To characterize the fidelity of the mRNA arising from dual vectors, HEK293 cells were infected with dual vectors and RNA extracted, reverse transcribed, and subjected to PCR utilizing primers binding upstream of the overlap region and in the bGH polyA signal region producing a 4.5 kb PCR fragment (FIG. 26A). An identically treated sample not containing reverse transcriptase was used as control for chromosomal DNA contamination. Plasmid containing the full-length MYO7A coding sequence was used as positive control for PCR. A preliminary screen of AAV-mediated MYO7A mRNA was performed by analyzing the pattern of fragment migration on agarose gel following restriction endonuclease digests with PpuMI and BglII (FIG. 26A). Identical banding patterns, consistent with the predicted pattern (PpuMI: 1591, 876, 556, 548, 541, 238, 168, 42, and 36 bp; BglII: 1335, 1074, 827, 583, 360, 272, and 146 bp), were observed following digests of amplicons from each dual-vector platform tested, indicating that no gross alterations (deletions/insertions) occurred as a consequence of either homologous recombination of vector pairs and/or RNA splicing (FIG. 26B). To further characterize the fidelity of the overlap region, a fragment containing the complete overlap area (1829 bp) was restricted and cloned into pUC57 (FIG. 26A). Sequencing results of 10 clones picked at random per vector platform revealed that the overlap region was 100% identical to the consensus/predicted MYO7A sequence (FIG. 26C). This indicated that, in the context of the simple overlap platform, homologous recombination was accurate. Additionally, in the context of trans-splicing vectors, accurate splicing occurred. Finally, for the AP hybrid vectors, a combination of accurate homologous recombination and/or splicing took place. To determine whether this protocol was capable of detecting aberrant sequence in reconstituted MYO7A, a sequence that contained either an insertion of a HindIII recognition site (TAGC) at position 2635 or a point mutation (T-C) at position 2381 was also generated.

MYO7A expression mediated by dual vectors in mouse retina. To investigate the expression of MYO7A from the two best performing dual-vector platforms in vivo, C57BL/6J mice were subretinally injected with 1×10¹⁰ vector genomes per eye of simple overlap and AP hybrid systems packaged in AAV8(733) and analyzed 4 weeks later by Western blot and immunohistochemistry. AAV8(733)-fAAV-MYO7A vector was also injected to provide a basis for comparison. To distinguish between endogenous MYO7A and exogenous expression mediated by vectors, sequence coding for an HA tag was added to the C′ terminus of the MYO7A cDNA in all constructs. Resulting retinas were immunostained for HA to reveal that fAAV vector along with both dual-vector platforms mediated expression of MYO7A in photoreceptors and RPE. A recent report concluded that simple overlap vectors were more efficient for gene transfer to the RPE than photoreceptors (Trapani et al., 2013). Simple overlap-mediated MYO7A expression was observed in both RPE and photoreceptors. In contrast to previous results showing “spotty” MYO7A expression mediated by AAV2-based simple overlap vectors (Lopes et al., 2013), it was found, when packaged in AAV8(733), that simple overlap vectors mediated MYO7A expression in the majority of RPE and photoreceptor cells. Photoreceptor degeneration/outer nuclear layer thinning was apparent in eyes injected with the AP hybrid vector system. Despite the observed degeneration, AP hybrid-mediated MYO7A was clearly detected in residual PR cell bodies and RPE and was sufficient to be detected by immunoblot. By Western blot analysis using HA antibody, simple overlap-mediated MYO7A was present in just detectable amounts. In contrast, fAAV-mediated protein levels were insufficient to be detected in this assay. Using an antibody against MYO7A, immunoblot of WT mouse retina revealed that both endogenous MYO7A and dual-vector-mediated, HA-tagged MYO7A migrated similarly.

Discussion

In this example, it was shown that dual AAV vectors with defined genetic payloads can be used to deliver a large transgene in vitro and in vivo. The initial experiments using the simplest of all dual-vector platforms revealed that efficiency of AAV2-based simple overlap vectors is proportional to the amount of 5′ and 3′ vectors used and that MYO7A expression mediated by this system increased as a function of incubation time in HEK293 cells. Next, three distinct dual-vector platforms were evaluated and compared to single, fragmented fAAV vector in vitro. All dual vectors analyzed drove higher levels of MYO7A expression than fAAV. Of all platforms tested, a hybrid vector system containing overlapping, recombinogenic sequence and splice donor/acceptor sites from the AP gene (AP hybrid) was the most efficient.

Regarding the specificity with which the dual-vector platforms express the correct-sized gene product, it was noted in vitro that trans-splicing and hybrid dual-vector platforms generated an additional band of lower molecular weight as detected by immunoblot (monoclonal antibody used was raised against the amino terminus MYO7A). The expression of this truncated product was much more pronounced for infections with 5′ vectors alone. What might account for this additional band? After entry into the host cell, the virus capsid is removed and the single-stranded DNA payload is released. The ITRs carried by the single strand serve as primer for DNA polymerases to produce a double strand. The resulting circular intermediates consist mainly of monomers that, over time, convert into multimeric concatemers through intermolecular recombination (Duan et al., 1998; Yang et al., 1999). The dual-vector systems created in this study utilize this strategy to achieve full-length protein expression. A limiting factor lies in the fact that the highly recombinogenic ITRs flanking the expression cassettes are identical in nature leading to a random recombination and consequently a random orientation of the vector parts relative to each other. This random recombination inevitably results in reduced efficiency because only concatemers that have the two vector parts in 5′-3′ orientation are able to express the full-length protein. This concatemerization over time is consistent with the observation that the amount of single-vector product is reduced in favor of the full-length protein when both 5′ and 3′ vectors are combined. Interestingly, the simple overlap system does not generate truncated product, even when only the 5′ vector is used for infections. In contrast to the trans-splicing and hybrid vectors, there is virtually no intervening sequence between the end of the MYO7A coding sequence and the right-hand ITR. It may be that splice donor sequences enhance the likelihood of truncated product through some as-yet-to-be-determined mechanism.

A number of strategies have been devised to overcome the issue of random concatemerization and thereby increase specificity as well as efficiency of these dual-vector platforms. First, the addition of a highly-recombinogenic sequence such as that used in the AP hybrid vector here has resulted in significantly increased protein expression compared with the trans-splicing system. Ghosh et al. (2011) provide a detailed analysis of the 270-bp AP sequence used in this study as well as other sequences derived from AP that direct recombination and lead to significant improvement over trans-splicing vectors. The finding that AP hybrid vectors are more efficient than trans-splicing vectors supports that the AP sequence directs at least some of the concatemerization events toward the proper orientation with recombination then occurring via this sequence or via the ITRs. Regardless, with more concatemers properly aligned, the AP hybrid system mediates a more-efficient expression of MYO7A. Another approach for directing concatemerization is the use of single-strand oligonucleotides that are capable of tethering the back end of the 5′ vector and the front end of the 3′ vector together (Hirsch et al., 2009). However, this strategy requires efficient delivery of the oligonucleotide to the nucleus of the target cells timed with the dual vectors. Finally, dual vectors utilizing mismatched ITRs can be used to direct concatemerization in a head-to-tail orientation (Yan et al., 2005), although the process may require further optimization of the AAV packaging machinery.

Notably, in this study, it was found that the sequence in the overlap region of all dual vectors tested in vitro was 100% identical to the consensus/predicted MYO7A sequence. This indicates that homologous recombination and/or splicing was accurate in each dual-vector platform.

Similar to the in vitro results, the highest levels of MYO7A expression was found in retinas of mice subretinally injected with AAV8-based AP hybrid vectors (as assessed by probing for HA on Western blot). Notably, no truncated proteins were evident in retinas expressing either simple overlap or AP-hybrid mediated MYO7A. The reason for this observed difference remains to be elucidated but may involve differences in the DNA repair machinery that mediate recombination in actively dividing cells versus post-mitotic photoreceptors/RPE (Hirsch et al., 2013). Dual-vector-mediated MYO7A-HA expression was observed in the photoreceptors and RPE of WT mice, locations where MYO7A is thought to have a functional role (Williams and Lopes, 2011). In eyes injected with AP hybrid vectors, marked thinning of the outer nuclear layer was observed. It has previously been shown that vector-mediated overexpression of MYO7A leads to retinal toxicity (Hashimoto et al. 2007). Taken together with the high efficiency of transduction observed in vitro for the AP hybrid platform, the most likely explanation for the observed pathology is excessive production of MYO7A. Despite the marked degeneration, significant amounts of AP hybrid-mediated, full-length MYO7A-HA were detected on Western blot. As high concentrations of vectors were used in these experiments, a simple solution to circumvent toxicity could be to reduce vector genomes injected or replace the strong, ubiquitous smCBA promoter with an endogenous or homologous promoter, and/or a promoter with attenuated strength. An alternative explanation for toxicity of the strong AP hybrid platform is expression of undesired products, like the observed protein expressed from the 5′ vectors alone in vitro. However, it was noted that only full-size MYO7A-HA was apparent on Western blot of the AP hybrid-treated retina.

With the goal of developing an AAV-based treatment for USH1B, animal models of this disease have provided an abundance of useful information. Similar to previous observations that fAAV-MYO7A and simple-overlap, dual-vectors were capable of restoring melanosome migration and opsin localization in the shaker1 mouse (Lopes et al., 2013), a recent study by an independent lab confirmed the usefulness of the vectors disclosed herein, when it was reported that they were capable of restoring the ultrastructural retinal phenotypes in the animal model. Notably, shaker1 mice lack retinal degeneration, and the severe functional abnormalities seen in USH1B patients (Liu et al., 1997). This fact renders in vivo analysis of therapeutic outcomes in the shaker1 retina problematic. Alternative animal models for evaluating a treatment for this devastating disease may be useful in adaptation of the present methods to human clinical use.

These results presented here also demonstrated that MYO7A can be efficiently expressed using dual-AAV-vector systems. The platforms containing overlapping elements, namely, the simple overlap system, and the AP hybrid system were both highly efficient. AP hybrid vectors showed the strongest expression of all systems tested, with little observable truncated protein in vitro and none observed in vivo. Simple overlap vectors showed good expression and were the most specific (no truncated protein products were observed) even when the 5′-only vector was used to infect cells. AAV has emerged as the preferred clinical vector and it efficiently transduces both photoreceptors and RPE. Because it has now been demonstrated that MYO7A sequence fidelity is preserved following recombination and/or splicing of dual-AAV-vector platforms and because only full-length MYO7A was detectable in mouse retinas injected with dual vectors, the dual-AAV-vector strategy presented herein represents a valid option for the treatment of retinal disorders associated with mutations in large genes such as USH1B.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

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It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

All references cited herein (including publications, patent applications and patents) are incorporated by reference to the same extent as if each reference was individually and specifically incorporated by reference, and was set forth in its entirety herein.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

All methods described herein can be performed in any suitable order, unless otherwise indicated herein, or unless otherwise clearly contradicted by context.

The use of any examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the invention using terms such as “comprising”, “having”, “including” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that “consists of”, “consists essentially of”, or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations may be applied to the compositions and/or methods disclosed herein, and/or to the steps or the sequence of steps of the methods described herein without departing from the concept, spirit and/or scope of the invention. More specifically, it will be apparent that certain agents that are chemically- and/or physiologically-related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 

1.-25. (canceled)
 26. A polynucleotide vector system comprising i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end of the polynucleotide, and between the inverted terminal repeats a promoter followed by a partial coding sequence that encodes an N-terminal part of a selected full-length polypeptide followed by a splice donor site and an intron, and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end of the polynucleotide, and between the inverted terminal repeats an intron and a splice acceptor site for the intron, and optionally followed by a partial coding sequence that encodes a C-terminal part of the selected full-length polypeptide, followed by a polyadenylation (pA) signal sequence, wherein the intron sequence in the first and second AAV vectors comprises a polynucleotide sequence that overlaps.
 27. The polynucleotide vector system of claim 26, wherein the polynucleotide sequence overlap in the intron sequence in the first and second AAV vectors is about 50 to about 500 nucleotides in length.
 28. The polynucleotide vector system of claim 26, wherein the promoter is selected from the group consisting of a chimeric CMV β actin (smcBA) promoter, a human myosin 7a gene-derived promoter, a cone transducin α (TαC) gene-derived promoter, a rhodopsin promoter, a cGMP-phosphodiesterase β-subunit promoter, a human rhodopsin kinase (hGRK1) promoter, a rod specific IRBP promoter, a RPE-specific vitelliform macular dystrophy-2 [VMD2] promoter, and combinations thereof.
 29. The polynucleotide vector system of claim 26, wherein the selected full-length polypeptide is encoded by a gene of about 5 Kb to about 10 Kb in length.
 30. The polynucleotide vector system of claim 26, wherein the selected full-length polypeptide is human myosin VIIa, or a functional fragment thereof.
 31. The polynucleotide vector system of claim 30, wherein the human myosin VIIa polypeptide comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8, or a functional fragment thereof.
 32. The polynucleotide vector system of claim 26, wherein the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:3, or a functional fragment or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:4, or a functional fragment or variant thereof.
 33. An isolated host cell comprising the polynucleotide vector system of claim
 26. 34. The isolated host cell of claim 33, wherein the cell is a photoreceptor cell, a cone cell, a rod cell, a retinal cell, or any combination thereof.
 35. A virus or an infectious viral particle comprising the first AAV vector polynucleotide or the second AAV vector polynucleotide of claim
 26. 